Patent Application
20240226010
This application includes a Sequence Listing submitted electronically in XML format. The XML copy of the Sequence Listing, created on Jan. 11, 2024, is named 21764-4-1-SeqList.xml and is 7,724 bytes in size. The XML copy of the Sequence Listing is expressly incorporated herein by this reference.
At the most basic level of design, the goal of a pharmaceutical agent is to target diseased tissue while preserving healthy tissue and cells. However, there are countless barriers to successfully targeting only diseased tissue in a system as complex as the human body. From macroscopic considerations of venous pathways to microscopic highly trafficked gateway of the lipid membrane, with each level of physiology specificity is crucial to reaching the desired target.
To address such issues, technology is now focusing the design of drug and gene delivery systems on nano- and micro-molecular scales. Protein and biopolymer-based carriers transport their delivery cargo through many different methods employing chemical conjugation, non-covalent association by electrostatic interactions, or physically packaging the therapeutic product.
Further, synthetic carriers are preferred over viral ones because they eliminate the risk of immunogenicity and mutagenesis, which are typically associated with viral-based systems. An optimal delivery system requires targeting specificity, efficiency in cellular and/or nuclear uptake, low cytotoxicity, and a modifiable platform to enable adaptation for different physiological environments.
An elastin-like peptide (ELP) is a genetically engineered biopolymer based on the sequence of human elastin (and therefore not recognized as foreign material by the body). It has a repeating sequence of Val-Pro-Gly-X-Gly (VPGXG) (SEQ ID NO:1), where the fourth guest residue X can be any amino acid except proline. ELP undergoes reversible inverse phase transition that occurs at a characteristic lower critical solution temperature (LCST), resulting in a change from a disordered hydrated polymer to a more structurally ordered coacervate. This LCST can be raised or lowered by modifying the ELP structure or by changing the polymer concentration and ionic strength present in solution. ELP's provision for LC ST modification and its ability to impart its inverse phase transition behavior to other proteins by fusion have led to its applications in drug and peptide delivery, protein purification, and tissue engineering.
Understanding ELP's inverse phase transition behavior is important for ELP's use in applications including drug delivery, gene transfection, diagnostics, and as biologically active coatings. While the solution behavior of ELP has been extensively studied, the interaction between the ELP and a surface has not.
Innovative approaches have used biocompatible and biodegradable ELP conjugated to drugs that are less toxic than free drug systemic delivery. Further, their delivery is assisted by the enhanced permeability and retention effect due to their thermally induced coacervation. Functional or therapeutic peptides can be easily added to ELP, either by chemical reaction, genetic engineering, or both, further improving their drug delivery and function.
One previously used synthetic carrier is branched polyethyleneimine (PEI), a cationic polymer, known for its high efficiency as a non-viral vector for cell transfection. PEI's positive charges on the primary amine groups allow it to complex with negatively charged phosphates of DNA and the anionic cell membrane for efficient cell uptake. However, because transfection efficiency increases with PEI molecular weight and its cationic nature enables gene delivery, this also makes PEI cytotoxic to cells. Cytotoxicity of PEI may possibly be improved by grafting it to higher molecular weight biocompatible polymers. However, PEIs with an attached hydrophilic polymer also form less-compact structures, exhibit less efficient aggregation, and have decreased ability to evade endosomal processing. Alternatively, attached hydrophobic biocompatible moieties to PEI have shown a decrease in cytotoxicity, greater compression of carrier/DNA complexes for enhanced cell uptake, and a drastic improvement of the transfection efficiency.
Previous studies have shown that ELP-PEI copolymers exhibit both the inverse phase transition behavior of ELP as well as cell biocompatibility. ELP-PEI copolymers could serve as a modifiable platform that combines ELP's capability for thermally induced self-aggregation and biocompatibility with the transfection efficiency and control of particle radius conferred by adding a block of PEI. ELP-PEI copolymers thus have potential as a biotherapeutic delivery agent whose dosage can be controlled by cross-linking particles at the desired nanometer or macromolecular size.
Accordingly, it would be an improvement in the art to provide a method by which the size of ELP-based polymers is controlled.
Exemplary methods disclosed herein are designed to improve the biocompatibility and transfection efficiency of elastin-like peptide (ELP)-containing particles by controlling the size of the particles. Such a method may include adding a polymer to an ELP in solution and then coupling the polymer to the ELP to form an ELP-polymer copolymer. The method may also include heating the ELP-polymer copolymer at or above a lower critical solution temperature (LCST) of the ELP to form macro- or micro ELP-containing particles of desired size. Finally, the method may include crosslinking a plurality of ELP-polymer copolymers to form particles of the desired size resistant to disaggregation below the LCST.
In an exemplary embodiment, the method of manufacturing macro- or micro ELP-containing particles of controlled size includes adjusting one or more of temperature, ion concentration, or pH of the solution of polymer and ELP to facilitate controlled particle formation of a desired size. The method can also comprise depositing the solution onto a hydrophilic surface and dehydrating the solution to form particles of a desired size and/or shape.
For example, the interaction between ELP and silica and how the ELP behaves in prolonged contact with silica above its LC ST was investigated after the evaporation of water. Specifically, the distribution, size, and maturation of the ELP coacervates, after prolonged exposure to a surface above the LCST, was investigated. The de-solvated state gives the ability to image the process using a scanning electron microscope (SEM), which would not be possible if water were present. It was found that ELP undergoes a complex rearrangement where it begins as a smooth film that eventually de-wets the silica to form a narrow distribution of particle sizes that dynamically grow and collapse over 24 hours.
ELP was shown to have a dynamic behavior while in contact with silica that shows similarities to viscoelastic polymer de-wetting. A combination of multiple factors was shown to contribute to this behavior, including the hydrophilicity of the silica preventing the adsorption of ELP, the formation of a salt layer between ELP and silica, and the ability of the silica and salt layer to hold on to minute amounts of water for prolonged periods. Particle size and distribution can be controlled by varying the time the ELP is raised above its LCST. This demonstrates the complex interactions that ELP can undergo when in contact with a hydrophilic surface and demonstrates that ELP-surface interactions could be used to create precisely defined particles and to create surfaces that dynamically rearrange in response to their environment.
The disclosed methods can also include adding a polymer block (e.g., PEI) to the terminal end of ELP, and allowing the particle radius as well as the LCST to be controlled by adjusting any combination of polymer concentration, ion concentration (e.g., from NaCl), and pH. The manipulation of the solution environment can therefore provide a method whereby ELP-containing particles are tailored for specific applications. The addition of the polymer block also provides the ability to crosslink the copolymers and achieve a stable particle radius after formation in harsh environments.
Additional features and advantages of the disclosure will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by the practice of the disclosure. The features and advantages of the disclosure may be realized and obtained by means of the instruments and combinations particularly pointed out in the appended claims. These and other features of the present disclosure will become more fully apparent from the following description and appended claims or may be learned by the practice of the disclosure as set forth hereinafter.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
In order to describe the manner in which the above recited and other advantages and features of the disclosure can be obtained, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments thereof, which are illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the disclosure and are not therefore to be considered to be limiting of its scope. The disclosure will be described and explained with additional specificity and detail through the use of the accompanying drawings in which:
All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.
All terms, including technical and scientific terms, as used herein, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs unless a term has been otherwise defined. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning as commonly understood by a person having ordinary skill in the art to which this disclosure belongs. It will be further understood that terms, such as those defined in commonly used dictionaries, should be interpreted as having a meaning that is consistent with their meaning in the context of the relevant art and the present disclosure. Such commonly used terms will not be interpreted in an idealized or overly formal sense unless the disclosure herein expressly so defines otherwise.
As used in the specification and claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a measurement” includes a plurality of separate measurements.
The embodiments disclosed herein should be understood as comprising/including disclosed components, and may therefore include additional components not specifically described. Optionally, the embodiments disclosed herein are essentially free or completely free of components that are not specifically described. That is, non-disclosed components may optionally be completely omitted or essentially omitted from the disclosed embodiments.
The numerical values set forth in the specification and examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from imprecisions associated with their respective testing measurements. Numeric values may optionally be modified by express use of the term “about” or “approximately”, which when expressly used mean±10%, ±5%, ±1%, or ±0.5% of the stated numeric value depending on context, significant digits, whether expressly defined as such, etcetera.
In the field of nanoparticulate drug delivery systems, the term “micelle” is understood to refer to a nano-sized structure formed from amphiphilic molecules that can self-assemble into a vesicle structure and that has a diameter of less than 100 nm (corresponding to the typical definition of a “nanoparticle”). Thus, a “particle” with a particle size (e.g., diameter) greater than 100 nm is not a “nanoparticle” or “micelle” and shall be understood to exclude “micelles” as that term is ordinarily understood by one skilled in the art. (See e.g., “Terminology for biorelated polymers and applications (IUPAC Recommendations 2012”, Pure Appl. Chem., Vol. 84, No. 2, pp. 380, 390, 392-394, 2012.)
The term “micro-sized particle” means a particle having a size in a range greater than 0.1 μm and up to 10 μm. The term “macro-sized particle” means a particle having a size greater than 10 μm. Micro- and macro-sized particles are not micelles according to the definitions used by those of skill in the art. (See id.)
The term “average” is used herein to refer to the “mean.” Mean particle sizes are reported herein based on the measurement method used. For measurements made using scanning electron microscopy (SEM), image analysis software can be used to determine the mean diameter. Means calculated from image analysis measurements will inherently be number-weighted. Thus, the term “mean diameter,” as used herein, refers to a number-weighted mean. For measurements made using dynamic light scattering (DLS), the standard output is radius of hydration (Rh), also known as the hydrodynamic radius. The skilled person will understand that reported Rh values are derived from intensity weighted size distributions, since DLS measures relative intensity of the light scattered by the particles versus a series of size classes. Accordingly, a reported mean Rh value represents the intensity weighted mean value (also known as the z-average). See ISO 13321 and ISO 22412. Unless indicated otherwise, particle size values disclosed herein as diameter measurements are number-weighted mean diameters, and particle size values disclosed herein as radii are intensity weighted mean radii.
Moreover, the hydrodynamic radius is defined as the radius of a sphere that diffuses at the same rate as the particle, regardless of the particular shape of the particle. Thus, the skilled person will understand that the hydrodynamic radius is, by definition, indifferent to particle shape.
The present disclosure is directed to methods for manufacturing polypeptide-based particles of controlled size. For example, the inventors have discovered that SynB1-ELP may exhibit dynamic behavior when disposed upon silica and show similarities to visco-elastic polymer de-wetting. A combination of multiple factors was shown to contribute to this behavior, including formation of a flat salt layer between ELP and silica and ability of the silica and salt layer to hold on to small amounts of water for prolonged periods. ELP particle size and particle size distribution can be controlled by varying the time ELP spends above the LCST. ELP-surface interactions may be used to create precisely defined ELP particles and to create surfaces that dynamically rearrange in response to their environment.
Exemplary embodiments are set forth employing methods and compositions for the production of novel macro-sized and micro-sized particles that combine the functionality of ELP and a polymer such as PEI. The inventors have discovered that by adding a polymer (e.g., PEI) block to the terminal end of ELP, the particle radius as well as LCST can be controlled by changing any combination of polymer concentration, ion (e.g., from NaCl) concentration, and pH.
The inventors have also observed that increasing ion concentration has the greatest impact on polymer behavior for ELP-PEI co-polymers, resulting in larger sizes of the particle radius. Similarly, increasing the polymer concentration may also have a demonstrated effect on both the particle radius and LCST of ELP-PEI co-polymers. Lastly, pH has a fine-tuning effect on both the particle radius and LCST of ELP-PEI co-polymers. While the molecular weight of the added PEI block does not have a significant impact on radii between the two ELP-PEI copolymers, molecular weight can however, determine how responsive the copolymer will be to the small changes in solution conditions and hence its ability to be tailored for specific applications.
Therefore, implementation of the present invention allow for methods by which ELP-PEI copolymers are coarsely tuned with salt to get within a particular window of LCST and particle radius, while polymer concentration and pH are be used as modulators for fine tuning of the copolymer properties.
In an exemplary embodiment of the present invention, ELP peptides may be coupled to a cargo therapeutic agent including, but not limited to, SynB1. The description related to the example SynB1 is also applicable to alternative therapeutic agents. SynB1, also known as SynB1 peptide, has the sequence RGGRLSYSRRRFSTSTGRA (SEQ ID NO:2). In some embodiments, the ELP sequence comprises at least 5 repeats to 160 repeats of the amino acid sequence VPGXG (SEQ ID NO:1), where X in the sequence VPGXG can be any amino acid except proline. In some embodiments, the method includes production of macro- and micro-particles comprising an ELP coupled to a therapeutic agent, small molecule, or drug used to deliver an effective amount of the agent or other active molecule to a biological system, either in vivo, in situ, or in vitro for effective control or treatment. The SynB1-ELP peptide can be genetically produced from prokaryotic or eukaryotic cells using the gene encoding the SynB1-ELP peptide.
In other embodiments, the peptide may be collected from the supernatants of expression media or cell extracts and purified using conventional purification methods known to those skilled in the art. Additionally, ELP may have the property of being thermally responsive. At or above a characteristic transition temperature (Tt), ELP can aggregate and precipitate, and when the temperature is lowered below the Tt, ELP can re-dissolve. Therefore, purification of ELP after expression in bacteria can include heating the bacterial lysate above the Tt and collecting ELP or ELP fusion proteins by centrifugation. Repeated centrifugations above and below the Tt can result in pure ELP. Purified SynB1-ELP peptide can be equilibrated into phosphate buffered saline and maintained as a stock below the LC ST until use for particle formation.
In some embodiments, the composition additionally or alternatively comprises a cell-penetrating peptide coupled to the ELP. Non-limiting examples of the cell-penetrating peptide are penetratin, TAT (GRKKRRQRRRPQ) (SEQ ID NO:3), SynB1, Bac, polyArg, MTS, Transportan, KLAK (SEQ ID NO:4), or pVEC. The composition may additionally or alternatively comprise a cellular or organ targeting peptide coupled to the ELP. Non-limiting examples of cellular targeting peptide are an antibody, a receptor binding factor or substrate, or carbohydrate containing motif. Non-limiting examples of organ targeting peptide are kidney targeting peptide, a placenta targeting peptide, or a brain targeting peptide. Non-limiting examples include coupling the drug to the ELP via using a genetic sequence encoding a drug binding domain at either terminus of the ELP protein that facilitates crosslinking of the drug or biochemical compound of interest to the ELP. An ELP protein comprising one or more cargo agents coupled thereto may be utilized in any of the embodiments disclosed herein.
An example method for forming micro- and macro-sized particles comprises applying solutions below the LC ST containing an ELP; or ELP linked to a cargo peptide, drug, small molecule, or other polymer; or an ELP that encapsulates a non-linked peptide, drug, small molecule, or other polymer; a buffer; and a salt concentration on a hydrophillic, hydrophobic, charged, or neutral surface, such as a fused silica media. Using the surface de-wetting process, the liquid is placed into a heated environment above the LCST for a period of time until the desired macro- and micro-sized particles and shapes are obtained. Factors for particle formation may govern protein adsorption, including the expulsion of water from the protein and surface, the ability of the protein to undergo conformational changes, and the rearrangement of charges in the outer layer of the protein to align with those on the surface.
The sequence of a particular ELP can be modified such that it is possible to generate chimeras of ELP fused to therapeutic proteins or peptides and/or to add reactive sites for attachment of therapeutic agents. Such ELP chimeras provide certain therapeutic advantages to the therapeutic agent, such as comparatively better stability, solubility, bioavailability, half-life, persistence, and/or biological action of the therapeutic proteinaceous component or attached small molecule drug.
The fused silica media can be prepared in a laminar flow hood by rinsing with acetone and then water to remove any loose or soluble debris. The silica media can be soaked, for example, in 10% weight solution of hydrofluoric acid to remove any remaining contaminates. Last, the media can be rinsed with water to remove residual hydrofluoric acid, rinsed with acetone, and placed in an oven until dry.
In some embodiments, the aggregation of macro- and micro-ELP particles, including those coupled to therapeutic agents, such as SynB1-ELP and like, are produced by varying the exposure of droplets on a hydrophilic surface for a period of time ranging from 0.5 hours to 24 hours at temperatures above the transition temperature. In some embodiments, the protein concentrations of the ELP or ELP-linked cargo range from 0.1 mg/mL to 10 mg/mL. The stability of the protein's structure in solution at the time of interaction with the hydrophilic surface allows for ELP aggregation. In some embodiments, the formation of larger ELP aggregates using a hydrophilic surface and exposure to a heated environment above the transition temperature forms particle sizes ranging from 0.10 μm to 120 μm in size, or from 0.5 μm to 120 μm in size, or from 1 μm to 120 μm in size, the foregoing referring to number-weighted mean diameters. Preferably, the particle shapes formed are spherical, hemispherical, or disc shaped. In yet other embodiments, SynB1-ELP's stability at temperatures above the phase transition, coupled with its low overall positive charge at neutral pH, prevents SynB1-Cys-ELP from adsorbing onto the hydrophilic silica substrate.
An ELP molecule can be coupled to a separate polymer molecule to form an “ELP-polymer copolymer.” An ELP can be coupled to the polymer in addition to or as an alternative to the one or more cargo agents disclosed above.
Some embodiments include using one or more of polymer concentration, pH, and/or ion concentration (e.g., from NaCl) to alter the LC ST properties and particle size of ELP/ELP-polymer co-polymers. PEI polymer sizes can range from 800 g/mol or smaller to 10,000 g/mol or larger. By way of example, ELP and PEI coupled co-polymers having sizes of MW=800 g/mol or 10,000 g/mol are referred to herein as ELP-PEI 800 and ELP-PEI 10K, respectively.
Some embodiments include a method for producing micro and macro-sized ELP and polymer particles having mean radii ranging from 200 nm to 1700 nm and transition temperatures ranging from 24° C. to 55° C. utilizing concentrations of ions (e.g., from NaCl) to control aggregate sizes and to induce transition temperatures. Polymers include negatively charged, positively charged, and neutral organic polymers, as well as polypeptide polymers such as collagen and fibrinogen.
Examples of organic polymers include: positively charged polymers, such as polyethyleneimine (PEI), polylysine, polyarginine, poly(diallyldimethyl ammonium chloride); negatively charged polymers such as polyacrylic acid, hyaluronic acid, poly(sodium styrene sulfonate), DNA; and neutral polymers such as polyethylene glycol, poly(vinyl pyrrolidone), poly(vinyl alcohol). Further, copolymers and amphiphilic polymers that contain fractions of two or more monomer types (positive, negative, or neutral) can be attached to the ELP.
In some embodiments, the concentration of salt (e.g., NaCl) ranges from 200 mM to 1.0 M. In yet other embodiments, increasing polymer concentration yields a higher maximum Rh and a slight decrease in LCST (or transition temperature (Tt)). In some embodiments, adjusting the pH of the ELP solution ranging from 200 mM to 1.0 M provides for fine-tuning of the aggregate size.
Some embodiments may include, for example, a first step of reacting ELP with N-hydroxysuccinimide in MES buffer, pH 6.2, and EDC for 15 minutes at 4° C. The second step includes reaction with PEI at pH 6.5, overnight at 4° C.
In some embodiments, stabilizing the micro and macro-sized particles includes crosslinking the ELP-polymer particles. Crosslinking allows for the retention of the particle sizes and shapes after they are formed, without the need for maintaining the negative environments in which they are formed (e.g., high salt concentrations). Crosslinking need not be dependent upon surface properties and is achieved with any chemistry reactive towards carboxylic acids, amines, and/or thiol groups such as: multi-functional aldehydes, trans-glutaminase, epoxide chemistry, carbodiimide chemistry, isocyanate chemistry, thiol-ene/yne chemistry, acrylates, methacrylates, and so forth. In some embodiments, the crosslinking reaction includes a reaction between amine groups and aldehyde and/or ketone groups to form imine groups. For example, where a polymer including amine groups (such as PEI) is coupled to the ELP, a multi-functional ketone and/or multi-functional aldehyde (e.g., glutaraldehyde) can be used as crosslinking agent.
Reactions to primary amine(s) present in an ELP-polymer copolymer can be achieved via reactions using aldehydes, isocyanates, isothiocyanates, sulfonyl chlorides, ketones, nitrous acid to form nucleophiles, electrophilic substitution of the nitrogen, carbodiimide chemistry, and anhydrides. Reactions with the carboxylic acid group(s) present in an ELP can be achieved by using isocyanates, isothiocyanates, Fischer esterification, nucleophilic reactions, electrophilic reactions, non-reversible esterification, thiols, alcohol groups, primary or secondary amines, and Grignard reactions. In at least some embodiments, however, the crosslinking reaction does not include the formation of amide bonds.
If cysteine residue(s) are present in an ELP-polymer copolymer, such ELP-polymer copolymers can self-crosslink through cysteine-cysteine reactions after the removal of chemicals (e.g., Dithiothreitol (DTT)) that specifically prohibit such reactions. In addition, reactions to the thiol group(s) on the cysteine residue(s) present in an ELP-polymer copolymer can be achieved by using the reaction chemistries of thiol to alkene, thiol to alkynes, thiol-thiol oxidation, glutathione oxidation, thiol-gold, thiol to metals, thiol deprotonation leading to a nucleophilic reaction, Michael additions, substitution reactions using Sn2 mechanisms to form thioethers, thiol to isocyanates or isothiocyanates, thiols to acid halides, and thiols to carboxylic acids.
Reactions to the alcohol group(s) present in an ELP-polymer copolymer can be achieved by reactions with isocyanates, isothiocyanates, condensation reactions, acid halides, esterification, etherification, and nucleophilic substitutions.
The preceding chemistries used for crosslinking can also be used to attach any second type of polymer to the ELP-polymer copolymer. For example, carbodiimide chemistry can be used to attach a natural or synthetic poly-amino acid such as polyarginine or polylysine to the carboxylic acid group of the ELP. It can also be used to attach synthetic, positively charged polymers such as PEI.
The primary amine group on ELP can be reacted to negatively charged polymers such as polyacrylic acid via a carbodiimide reaction. Natural or synthetic neutral polymers such as polyethylene glycol can be attached via an isocyanate reaction or through an aldehyde reaction if they are pre-functionalized with an amine group.
Purification of crosslinked particles can be performed through known methods for small molecule removal, such as dialysis, filtration, and low speed centrifugation.
In at least one embodiment, the ELP or ELP-PEI copolymer micro and macro-sized particles are dissolved in water, and the appropriate environmental conditions are adjusted to achieve a desired size. ELP or ELP-PEI copolymer particles may also be heated above the transition temperature, and 10-fold concentration of glutaraldehyde may be added to the solution. The mixture may be reacted for 1 hour and, afterwards, particles may be purified by utilizing the inverse transition temperature of the ELP.
In some embodiments, the crosslinking reaction is carried out such that the polymer portions of the ELP-polymer copolymers are crosslinked to a greater degree than the ELP portions of the ELP-polymer copolymers. For example, the crosslinking reaction can be carried out such that the polymer portions of the ELP-polymer copolymers are substantially the only portions of the ELP-polymer copolymers that are crosslinked, while the ELP portions of the ELP-polymer copolymers remain substantially non-crosslinked. This can beneficially minimize interference with the temperature-dependent response functionality of the ELP segment of the ELP-polymer copolymer. As disclosed herein, the temperature-dependent response of the ELP-based particles can be beneficially utilized in a controlled manner to control particle size.
In some embodiments, the neat ELP and ELP-polymer copolymer mixtures are formulated with a molar ratio of ELP to ELP-polymer of 70:30 to 99:1, or 80:20 to 98:2, or 90:10 to 97:3, or 93:7 to 96:4, such as about 95:5, or a range with any two of the foregoing ratios as endpoints. ELP to ELP-polymer copolymer ratios within the foregoing ranges were found to beneficially balance effective crosslinking with biocompatibility, particularly when the polymer utilized in the ELP-polymer copolymer has some level of cytotoxicity when used in excess (e.g., as is the case with PEI). For example, ELP-PEI copolymers having molar ELP to ELP-PEI ratios within the foregoing ratios exhibited the most uniform size distribution of mammalian spheroidal cell aggregates when the ELP/ELP-PEI copolymer mixtures were coated onto culture surfaces.
In some embodiments, the particles resulting from the crosslinking reactions include crosslinking throughout the volume of the individual particles. That is, crosslinking is not necessarily confined to the outer shells or inner cores of the particles, but is instead present throughout the volume of the particles. For example, crosslinking may be substantially uniformly distributed throughout the volume of the resulting particles.
The ELP-cargo peptide selected for the macro- and micro particle formation was a SynB1-Cys-ELP peptide construct. SynB1-Cys-ELP peptide was prepared from a plasmid containing a repeat structure of MRFFRLSYSRRRFSTSTGRCGPG (SEQ ID NO:5) (VPG[V5G3A2]G)150WP encoding a peptide of MW=61,739 daltons. The plasmid was expressed by cloning into a pET25b vector and transformed into BLR (DE3) Escherichia coli. The overexpressed polypeptide was collected from the transformation media and purified by four rounds of thermo-cycling to a translucent pellet. Samples were equilibrated into PBS (phosphate buffered saline (150 mM NaCl, 2.7 mM KCl, 20 mM Na2HPO4, 1.8 mM KH2PO4)), by dialysis using a Slide-A-Lyse MINI Dialysis device purchased from Thermo Fisher Scientific with a 3,500 molecular weight cut-off and a particle size of less than about 20 nm.
Fused silica media selected as the substrate for ELP-cargo peptide particle deposition were 1.5-inch diameter disks obtained from Thorlabs. Media disks were cleaned by a standard cleaning procedure for fused silica in a laminar flow hood entailing first rinsing the substrates with acetone and then water to remove any loose or soluble debris. The disks were soaked in a 10% weight per volume solution of hydrofluoric acid for 5 minutes to remove any remaining contaminates. Afterward, the substrates were rinsed repeatedly with water to remove any acid, rinsed with acetone, and dried in an oven at 50° C. for 12 hours.
In preparation of ELP-cargo peptide particle formation, a stock of SynB1-Cys-ELP stored in PBS was used to make three increasing concentrations of SynB1-Cys-ELP: 0.8 mg/mL, 1.5 mg/mL, and 3 mg/mL. A 10 μL aliquot of SynB1-Cys-ELP solution was placed as a droplet onto a fused silica disk. The silica disk was subsequently placed into a 60-mm cell culture plate to prevent any accidental contamination from dust and incubated in a Thermo Lindberg/Blue M digital oven (Thermo Scientific, Waltham, MA) set at 50° C. Samples of the droplets at different time points were analyzed using different measurements over a 24-hour incubation period to visualize the surface mediated ELP behavior.
Several measurement methods were used to evaluate the properties of the surface aggregated ELP-cargo particles formed, including electron microscopy, chemical composition, DLS, atomic force microscopy, and FT-IR spectroscopy. DLS was used to study the size of the coacervates as they partition between the two phases. Though limited by the size of particles it can measure, DLS is able to show the formation of coacervated droplets with radii of hydration (Rh) up to several μm. DLS was performed using a DynoPro NanoStar instrument (Wyatt Technology, Santa Barbara, CA) equipped with Dynamics V7 version 7.19 software. SynB1-Cys-ELP droplet samples corresponding to concentrations of 0.8 mg/mL, 1.5 mg/mL, and 3 mg/mL were centrifuged for 3 min at 5° C. at 50,000 rpm in a Beckman Optima TLX ultracentrifuge (Palo Alto, CA). A volume of 10 μL sample was loaded directly from the centrifuge tube into a DLS cuvette and allowed to equilibrate for 15 min inside the instrument. The experiments were conducted with a ramp rate of 0.20° C./min from 20 to 50° C.
A microscopy method employed SEM measurement of particle size using a Leica EM ACE600 coating system (Germany) that vented with argon was used to sputter-coat the samples with a 4-nm thick gold-palladium coating to prevent charge buildup when imaging. A Zeiss Supra 40 electron microscope (Germany) using an SE2 Everhart-Thornley detector, an aperture size of 30 and an accelerating voltage of 3 keV was used for imaging the samples. Image analysis was performed using ImageJ version 1.51k (NIH). A calibrated scale was based upon measuring the scale bar on the SEM image by the ImageJ software, after which the particle diameters were measured by using the elliptical tool to outline individual particles. The minimum sample size of 50 particles established an accurate representation of the population. The particle analysis was reported as the mean diameter ±95% confidence interval. The particle sizes of the macro- and micro-sized particles are shown in
A chemical composition of samples was mapped using Energy Dispersive X-ray Spectroscopy (EDS) from EDAX Octane Plus (AMETEK Mahwah, NJ) that was coupled to the SEM described above. The accelerating voltage of the EDS was set to 5 keV for EDS to maximize the number of chemical elements that were detected and to minimize sample charging. EDAX Team software version 6.43 was used to collect and analyze the data.
Atomic Force Microscopy images were obtained using a Bruker Bioscope Catalyst AFM (Billerica, MA). Select samples were imaged in the large amplitude Scanasyst® mode with a Scanasyst-air probe at a rate of 0.250 Hz. Images were analyzed using Gwyddion software version 2.64.
Attenuated Total Reflectance (ATR) Fourier Transform-Infrared (FT-IR) Spectroscopy was performed using Perkins-Elmer Spectrum 100 FT-IR spectrometer (Waltham, MA) with a universal attenuated total reflectance accessory equipped with a diamond crystal and a fixed beam incidence angle. Independent samples were prepared by placing a volume of 10 μL at 4° C. onto silica disks at room temperature and subsequently incubating them at 50° C. The samples were placed into a portable desiccator for transport to minimize adsorption of atmospheric water and were placed onto the ATR crystal without any additional preparation. All spectra were collected and analyzed using Spectrum software version 6.0.2. A background spectrum was taken to eliminate the presence of noise caused by moisture and carbon dioxide from the background environment. The samples were then placed on the crystal and secured via a pressure clamp at 110 psi. Sixteen scans were taken in the spectral range of 4000 cm−1 to 650 cm−1 with a spectral resolution of 4 cm−1. The scans were plotted as transmission (%) versus wavenumber (cm−1).
DLS measurements showed that the three concentrations of ELP exhibited a concentration dependent LCST from 36 to 39° C. The DLS data of the three concentrations showed that coacervates of increasing Rh began to form above the corresponding LCST (
The SEM analysis of SynB1-ELP coacervate formation on silica surface was performed to examine the time dependent interaction of ELP upon prolonged contact with silica above its LCST. It was observed that after de-solvation, upon contact with silica media, the ELP formed into large spherical particles (denoted by arrows) between 2 and 6 hours that dynamically grew and collapsed over time and grew again into larger particles (
In
As a general trend, all three concentrations (0.8, 1.5, and 3 mg/mL, respectively) exhibited a growth in mean particle diameter from 0.5 hours (1.26±0.07 μm, 2.00±0.01 μm, 2.55±0.13 μm) to 2 hours (2.87±0.82 μm, 3.25±0.36 μm, 4.36±0.44 μm). The diameters leveled off at 2.5 hours for all three concentrations (3.29±0.77 μm, 3.23±0.39 μm, 4.68±0.43 μm). In between, the 1.5 and 3 mg/mL samples drastically increased in size at 3.5 hours (14.89±2.50 μm, 15.88±6.20 μm). By the 5-hour time point all three concentrations showed a large mean particle diameter (18.11±2.4 μm, 14.54±3.80 μm, 24.04±6.40 μm). After 6 hours (8.24±1.90 μm, 7.73±0.58 μm, 8.89±0.81 μm), the mean diameters dropped until 9 hours to their lowest measured values (1.10±0.16 μm, 1.29±0.17 μm, 1.27±0.13 μm). The particle diameters surged to higher values at 12 hours (12.34±2.30 μm, 14.46±4.30 μm, 11.91±4.04 μm) and by 24 hours the 1.5 and 3 mg/mL sample particles collapsed onto the surface of the substrate and form disks, while the 0.8 mg/mL sample continued to show particles with a small mean diameter of 1.78±0.26 μm.
EDS imaging was used to map the elements present on the sample surface, specifically to identify the chemical composition of the larger raised surface structures in
SEM images showed a crystalline NaCl layer had formed at the 0.5-hour time point (
In
Atomic Force Microscopy (AFM) is an effective method for imaging the coacervate particles because of its ability to obtain high magnification images in a fluid environment at various temperatures using a heated stage. Solution-based AFM is only useful after the coacervates have settled on the surface of the container because the size of the coacervates floating in solution are too large and scatter the laser light used to image them. Therefore, while SEM is useful at imaging objects, ultimately the technique can only provide two-dimensional images of three-dimensional objects and offers no way to quantify whether an object is truly raised off the surface.
In
In other examples, as a proof-of-principal, SynB1-ELP was conjugated to doxorubicin for cancer treatment using a cleavable linker having a cysteine residue coupled to doxorubicin via a disulfide bond, as described in U.S. Pat. No. 8,252,740 (incorporated herein by reference).
The ELP solution was placed onto the substrate at 4° C., a temperature where the ELP was still hydrated by the surrounding water, and the individual protein molecules remained isolated from each other, as can be seen in the DLS results (
As the sample was elevated in temperature, the ELP was forced to coalesce above its LCST as the coacervates form, and they sedimented to the bottom of the droplet, where they destabilized as they encounter the substrate. However, because the surface of the silica substrate was hydrophilic in nature, it retained the water molecules bound at the interface as indicated by FT-IR (
The 0.5-hour time point shows that ELP began to contract together exposing the NaCl layer. By doing so it created a void in the film as the ELP began to accumulate at the outer edge of the holes near the exposed salt. This is also a feature exhibited by polymers in a metastable state, typically during an annealing process where they are below their glass transition temperature (Tg). This metastable state allows for partial wetting of the substrate without a spontaneous self-association of the polymer. If annealing of a metastable polymer continues above its Tg, it exhibits spontaneous de-wetting of the substrate and forms droplets. While Tg is not known to occur in ELP, a similar phenomenon is seen at the 1-hour time point for all three concentrations (
At 3.5 hours, larger particles attached to the protein bands started to form for the 1.5 and 3 mg/mL samples. The formation of these larger particles is attributed to coalescence of the rim structures due to Plateau-Raleigh instability as the rim retracted inward. This instability occurs from a liquid's innate ability to minimize its surface area when in contact with a surface that it dislikes. The images indicate the Raleigh instability incurred by the ELP is characteristic to that of a viscoelastic polymeric solution due to the formation of rim structures and smaller size Goutte satellite droplets that are present between the rims and coacervates (
At the 6 hours-time point, the ELP again spontaneously retracted from the surface for all three concentrations, forming numerous small non-uniform particles. The larger coacervates observed at the 3.5 and 5-hour time points began to destabilize and collapse downward onto themselves (
The 6-hour time point ends particle growth for all three sample concentrations and a combination of collapsed structures formed a film. In
ELP (MW=17,000 Da), produced from genetically engineered E. coli, was coupled to PEI (Polysciences) having sizes of MW=800 g/mol or 10,000 g/mol in a two-step reaction, as shown in
Based on preliminary studies with ELP-PEI 10K coatings for the culturing of pre-adipocytes, a ratio of 1.2:98.8 ELP-PEI 10K to ELP was selected for testing. An equivalent weight percent of ELP-PEI 800 (15:85 ELP-PEI 800 to ELP) to that of ELP-PEI 10K was used to observe the difference the polymers exhibited given the selected environment. Because the regular repeat structure (NH2—[CH2]) of PEI and the large amount of amide groups present in ELP, it was very difficult to distinguish ELP-PEI 800 and ELP-PEI 10K copolymers with traditional techniques such as FT-IR, as shown in
A new protocol was developed to measure the efficiency of the reaction using an O-phthlaladehyde (OPA) assay system. The reaction conversion was determined by labeling the copolymer with OPA (Thermo Sci), after which the fluorescence was measured and compared to a previously generated standard curve (generated by first measuring the fluorescence of different combinations of ELP to non-reacted PEI (1:0, 1:0.05, 1:0.10, 1:0.15, etc.) at five concentrations (0.1, 0.2, 0.3, 0.4, 0.5 mg/mL)). The protocol showed that while the increased amination due to PEI conjugation increased the OPA fluorescence, the PEI fluorescence was somewhat masked by ELP at lower percent conjugations. This is an important finding that helps to more accurately estimate an unknown percent conjugation. However, as shown in
A Dynapro Nanostar DLS instrument (Wyatt Tech) was used to determine aggregation sizes of ELP-PEI 800 and ELP-PEI 10K copolymers in aqueous solutions by altering their concentration (0.1, 0.17, 0.3 mg/mL), solution pH (3, 7, 10), and NaCl concentration (0, 0.2, 1 M NaCl). For crosslinked particles, they were incubated at 4° C. for 1 hour and subsequently DLS was performed at a temperature below the transition temperature at 20° C. (See below).
The following studies were performed is determine the ELP phase transition properties when fused to two molecular weights of PEI, 800 g/mol and 10,000 g/mol, and the influence on the coacervate size by environmental factors including PEI concentration, pH, and NaCl concentration.
In the absence of NaCl, DLS measurements on ELP-PEI 800 showed a multiple modal transition curve, as shown in
Similarly, in absence of NaCl ELP-PEI 10K exhibited a more complex, tri-modal transition curve with an onset of 35° C. and a radius of hydration (Rh) value of 550 nm (
In the next set of studies, the effect of NaCl concentration on the Rh of ELP-PEI 800, and neat ELP as shown in
Crosslinking of the ELP particles was made possible by the addition of a copolymer such as PEI and allowed for the retention of the particle sizes and shapes, (including spherical and disc shapes), after they are formed while minimizing or removing the need for the negative environments needed to maintain them (e.g. high NaCl concentrations).
The ELP-PEI copolymer solution samples were heated above their respective LCST in an oven. The LCSTs ranged from 22 to 37° C. Glutaraldehyde was then added (10:1 molar ratio of aldehyde to ELP) and allowed to react with the ELP-PEI copolymer particles in the oven for 15 minutes. The samples underwent a thermo-cycling purification in a temperature-controlled centrifuge. For determining aggregation sizes of the crosslinked ELP-PEI copolymers and to ensure the crosslinking was successful the samples were cooled to 4° C., and DLS was performed at a static temperature of 20° C., which is below the LCST of the non-crosslinked particles.
In
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other aspects of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.
This application is a continuation-in-part of U.S. patent application Ser. No. 16/432,879, filed Jun. 5, 2019, which claims the benefit of U.S. Provisional Application No. 62/680,828, filed Jun. 5, 2018, and U.S. Provisional Application No. 62/688,677, filed Jun. 22, 2018, which are incorporated herein by reference in their entirety.
This invention was made with government support under Grant Number EB02006 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 20240130971 A1 | Apr 2024 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62680828 | Jun 2018 | US | |
| 62688677 | Jun 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16432879 | Jun 2019 | US |
| Child | 18373749 | US |
