PARTICLES COMPRISING PROTEINS ENCAPSULATED IN A POROUS FRAMEWORK AND METHODS OF USING THEREOF
20250368757 ยท 2025-12-04
Inventors
Cpc classification
C07K17/04
CHEMISTRY; METALLURGY
A61K9/5146
HUMAN NECESSITIES
International classification
C07K17/14
CHEMISTRY; METALLURGY
C07K17/04
CHEMISTRY; METALLURGY
Abstract
Disclosed are methods for producing matrix-encapsulated proteins, including matrix-encapsulated hemoglobin. Also provided are pharmaceutical compositions comprising a matrix-encapsulated hemoglobin, as well as methods of using thereof to treat hypoxia, cyanide poisoning, hydrogen sulfide poisoning, and/or azide poisoning.
Claims
1. A method for producing a population of matrix-encapsulated protein particles, the method comprising: (a) combining a first framework precursor, a second framework precursor, and a protein to form a reactant mixture; (b) incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; and (c) separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration; wherein the matrix-encapsulated protein particles comprise a protein encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor.
2. The method of claim 1, wherein the population of matrix-encapsulated protein particles has an average particle size, as determined by electron microscopy, of less than 200 nm, such as less than 180 nm, less than 160 nm, less than 140 nm, less than 120 nm, less than 100 nm, or less than 80 nm; or wherein the population of matrix-encapsulated particles has an average particle size of at least 500 nm, at least 750 nm, at least 1 micron, at least 1.5 microns, at least 2 microns, at least 2.5 microns, at least 5 microns, at least 10 microns, at least 20 microns, at least 30 microns, at least 40 microns, at least 50 microns, or at least 100 microns.
3. The method of claim 1, wherein the method exhibits an encapsulation efficiency, measured by the fraction of the mass of the protein in the resulting matrix-encapsulated protein over the total mass of protein initially charged in the reactant mixture, of at least 80%, such as at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, or at least 96%.
4. The method of claim 1, wherein the matrix-encapsulated protein particles have a zeta potential of less than 5 mV, such as of less than 6 mV, less than 7 mV, less than 8 mV, less than 9 mV, less than 10 mV, less than 11 mV, less than 12 mV, less than 13 mV, less than 14 mV, or less than 15 mV.
5. The method of claim 1, wherein the population of particles has a PDI of less than 0.100, such as less than 0.095, such as less than 0.090, less than 0.085, less than 0.080, less than 0.075, or less than 0.070.
6. The method of claim 1, wherein the porous framework comprises a metal-organic framework (MOF), metal-inorganic framework (MIF), and/or covalent-organic framework (COF).
7. (canceled)
8. The method of claim 1, wherein the first framework precursor comprises a metal salt and the second framework precursor comprises a ligand.
9. The method of claim 1, wherein the porous framework comprises a zeolitic imidazolate framework (ZIF).
10. (canceled)
11. The method of claim 1, wherein the first framework precursor comprises a Fe salt, a Co salt, a Cu salt, a Zn salt, or a combination thereof.
12. The method of claim 1, wherein the second precursor comprises a ligand selected from the group consisting of imidazoles and derivatives such as 2-methylimidazole, 2-ethylimidazole, 4-azabenzimidazole, benzimidazole, nitroimidazole, 2-chloroimidazole, and the like; carboxylic acids and derivatives such as 1,4-benzenedicarboxylic acid, 1,3,5-benzene tricarboxylic acid, imidazole carboxaldehyde, 2-aminobenzimidazolate, the like, or any combination thereof.
13. The method of claim 1, wherein the first framework precursor and the second framework precursor are present in the reactant mixture at a molar ratio of from 1:1 to 75:1, such as from 1:1 to 60:1, from 1:1 to 30:1, or from 15:1 to 30:1.
14. The method of claim 1, wherein the protein is selected from the group consisting of conalbumin, albumin, hemoglobin, haptoglobin, hemopexin, transferrin, methemoglobin, ovalbumin, -chymotrypsinogen A, -chymotrypsin, trypsin, trypsinogen, -lactoglobulin, myoglobin, -lactalbumin, lysozyme, ribonuclease A, or cytochrome c, a recombinant version thereof, or a combination thereof.
15. (canceled)
16. (canceled)
17. (canceled)
18. (canceled)
19. (canceled)
20. The method of claim 1, wherein the reactant mixture further comprises an etching agent, a chelating agent, or a combination thereof.
21. The method of claim 20, wherein the etching agent is comprises hydrofluoric acid (HF), ammonium fluoride (NH.sub.4F), the acid salt of ammonium fluoride (NH.sub.4HF.sub.2), sodium hydroxide (NaOH), nitric acid (HNO.sub.3), hydrochloric acid (HCl), hydroiodic acid (HI), hydrobromic acid (HBr), boron trifluoride (BF.sub.3), sulfuric acid (H.sub.2SO.sub.4), acetic acid (CH.sub.3COOH), formic acid (HCOOH), phosphoric acid (H.sub.3PO.sub.4), or any combination thereof.
22. The method of claim 1, wherein the chelating agent comprises ethylenediaminetetraacetic acid (EDTA) or a derivative thereof.
23. The method of claim 1, where step (c) comprises filtering the reactant mixture comprising the matrix-encapsulated protein particles by ultrafiltration against a filtration membrane, thereby forming a retentate fraction comprising matrix-encapsulated protein particles having a molecular weight above a cutoff value and a permeate fraction comprising unencapsulated protein and other impurities having a molecular weight of less than the cutoff value.
24. (canceled)
25. (canceled)
26. (canceled)
27. A pharmaceutical composition comprising a population of matrix-encapsulated hemoglobin particles prepared using the method defined by claim 1.
28. A method of treating hypoxia in a subject comprising administering the subject a therapeutically effective amount of the composition defined by claim 27.
29. (canceled)
30. A method for producing a population of matrix-encapsulated protein particles, wherein the protein comprises methemoglobin or polymerized methemoglobin, the method comprising: (a) combining a first framework precursor, a second framework precursor, and hemoglobin or polymerized hemoglobin to form a reactant mixture; (b) incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; (c) contacting the population of matrix-encapsulated protein particles with an oxidizing agent under conditions effective to convert the hemoglobin or polymerized hemoglobin to methemoglobin or polymerized methemoglobin; and (d) separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration; wherein the matrix-encapsulated protein particles comprise a protein encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor.
31-34. (canceled)
35. A method for producing a population of matrix-encapsulated protein particles, wherein the protein comprises methemoglobin or polymerized methemoglobin, the method comprising: (a) contacting hemoglobin or polymerized hemoglobin with an oxidizing agent under conditions effective to convert the hemoglobin or the polymerized hemoglobin to methemoglobin or polymerized methemoglobin; (b) combining a first framework precursor, a second framework precursor, and the methemoglobin or the polymerized methemoglobin to form a reactant mixture; (c) incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; and (d) separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration; wherein the matrix-encapsulated protein particles comprise methemoglobin or polymerized methemoglobin encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor.
36-38. (canceled)
Description
DESCRIPTION OF DRAWINGS
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[0027] Figure SA shows the O.sub.2 equilibrium curves for bHb and ZIF-8P-Hb nanoparticles in comparison to bHb, RBCs, and various types of HBOCs in the literature. Lines represent the mean from all batches.
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DETAILED DESCRIPTION
[0043] A number of embodiments of the disclosure have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the invention. Accordingly, other embodiments are within the scope of the following claims.
Definitions
[0044] To facilitate understanding of the disclosure set forth herein, a number of terms are defined below. Unless defined otherwise, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.
General Definitions
[0045] The term comprising and variations thereof as used herein is used synonymously with the term including and variations thereof and are open, non-limiting terms. Although the terms comprising and including have been used herein to describe various embodiments, the terms consisting essentially of and consisting of can be used in place of comprising and including to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing quantities of ingredients, reaction conditions, geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches.
[0046] As used in this specification and the following claims, the terms comprise (as well as forms, derivatives, or variations thereof, such as comprising and comprises) and include (as well as forms, derivatives, or variations thereof, such as including and includes) are inclusive (i.e., open-ended) and do not exclude additional elements or steps. For example, the terms comprise and/or comprising, when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. Accordingly, these terms are intended to not only cover the recited element(s) or step(s), but may also include other elements or steps not expressly recited. Furthermore, as used herein, the use of the terms a, an, and the when used in conjunction with an element may mean one, but it is also consistent with the meaning of one or more, at least one, and one or more than one. Therefore, an element preceded by a or an does not, without more constraints, preclude the existence of additional identical elements.
[0047] The use of the term about applies to all numeric values, whether or not explicitly indicated. This term generally refers to a range of numbers that one of ordinary skill in the art would consider as a reasonable amount of deviation to the recited numeric values (i.e., having the equivalent function or result). For example, this term can be construed as including a deviation of +10 percent of the given numeric value provided such a deviation does not alter the end function or result of the value. Therefore, a value of about 1% can be construed to be a range from 0.9% to 1.1%. Furthermore, a range may be construed to include the start and the end of the range. For example, a range of 10% to 20% (i.e., range of 10%-20%) can includes 10% and also includes 20%, and includes percentages in between 10% and 20%, unless explicitly stated otherwise herein.
[0048] It is understood that when combinations, subsets, groups, etc. of elements are disclosed (e.g., combinations of components in a composition, or combinations of steps in a method), that while specific reference of each of the various individual and collective combinations and permutations of these elements may not be explicitly disclosed, each is specifically contemplated and described herein.
[0049] Ranges can be expressed herein as from about one particular value, and/or to about another particular value. By about is meant within 5% of the value, e.g., within 4, 3, 2, or 1% of the value. When such a range is expressed, another aspect includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent about, it will be understood that the particular value forms another aspect. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It is also understood that there are a number of values disclosed herein, and that each value is also herein disclosed as about that particular value in addition to the value itself. For example, if the value 10 is disclosed, then about 10 is also disclosed.
[0050] As used herein, the terms may, optionally, and may optionally are used interchangeably and are meant to include cases in which the condition occurs as well as cases in which the condition does not occur. Thus, for example, the statement that a formulation may include an excipient is meant to include cases in which the formulation includes an excipient as well as cases in which the formulation does not include an excipient.
[0051] Administration to a subject includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation, via an implanted reservoir, parenteral (e.g., subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intraperitoneal, intrahepatic, intralesional, and intracranial injections or infusion techniques), and the like. Concurrent administration, administration in combination, simultaneous administration or administered simultaneously as used herein, means that the compounds are administered at the same point in time or essentially immediately following one another. In the latter case, the two compounds are administered at times sufficiently close that the results observed are indistinguishable from those achieved when the compounds are administered at the same point in time. Systemic administration refers to the introducing or delivering to a subject an agent via a route which introduces or delivers the agent to extensive areas of the subject's body (e.g. greater than 50% of the body), for example through entrance into the circulatory or lymph systems. By contrast, local administration refers to the introducing or delivery to a subject an agent via a route which introduces or delivers the agent to the area or area immediately adjacent to the point of administration and does not introduce the agent systemically in a therapeutically significant amount. For example, locally administered agents are easily detectable in the local vicinity of the point of administration but are undetectable or detectable at negligible amounts in distal parts of the subject's body. Administration includes self-administration and the administration by another.
[0052] As used here, the terms beneficial agent and active agent are used interchangeably herein to refer to a chemical compound or composition that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, i.e., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, i.e., prevention of a disorder or other undesirable physiological condition. The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, isomers, fragments, analogs, and the like. When the terms beneficial agent or active agent are used, then, or when a particular agent is specifically identified, it is to be understood that the term includes the agent per se as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, conjugates, active metabolites, isomers, fragments, analogs, etc.
[0053] A decrease can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.
[0054] Inhibit, inhibiting, and inhibition mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.
[0055] Inactivate, inactivating and inactivation means to decrease or eliminate an activity, response, condition, disease, or other biological parameter due to a chemical (covalent bond formation) between the ligand and a its biological target.
[0056] By reduce or other forms of the word, such as reducing or reduction, is meant lowering of an event or characteristic (e.g., tumor growth). It is understood that this is typically in relation to some standard or expected value, in other words it is relative, but that it is not always necessary for the standard or relative value to be referred to. For example, reduces tumor growth means reducing the rate of growth of a tumor relative to a standard of a control.
[0057] As used herein, the terms treating or treatment of a subject includes the administration of a drug to a subject with the purpose of preventing, curing, healing, alleviating, relieving, altering, remedying, ameliorating, improving, stabilizing or affecting a disease or disorder, or a symptom of a disease or disorder. The terms treating and treatment can also refer to reduction in severity and/or frequency of symptoms, elimination of symptoms and/or underlying cause, prevention of the occurrence of symptoms and/or their underlying cause, and improvement or remediation of damage. In particular, the term treatment includes the alleviation, in part or in whole, of the symptoms of coronavirus infection (e.g., sore throat, blocked and/or runny nose, cough and/or elevated temperature associated with a common cold). Such treatment may include eradication, or slowing of population growth, of a microbial agent associated with inflammation.
[0058] By prevent or other forms of the word, such as preventing or prevention, is meant to stop a particular event or characteristic, to stabilize or delay the development or progression of a particular event or characteristic, or to minimize the chances that a particular event or characteristic will occur. Prevent does not require comparison to a control as it is typically more absolute than, for example, reduce. As used herein, something could be reduced but not prevented, but something that is reduced could also be prevented. Likewise, something could be prevented but not reduced, but something that is prevented could also be reduced. It is understood that where reduce or prevent are used, unless specifically indicated otherwise, the use of the other word is also expressly disclosed. For example, the terms prevent or suppress can refer to a treatment that forestalls or slows the onset of a disease or condition or reduced the severity of the disease or condition. Thus, if a treatment can treat a disease in a subject having symptoms of the disease, it can also prevent or suppress that disease in a subject who has yet to suffer some or all of the symptoms. As used herein, the term preventing a disorder or unwanted physiological event in a subject refers specifically to the prevention of the occurrence of symptoms and/or their underlying cause, wherein the subject may or may not exhibit heightened susceptibility to the disorder or event. In particular embodiments, prevention includes reduction in risk of coronavirus infection in patients. However, it will be appreciated that such prevention may not be absolute, i.e., it may not prevent all such patients developing a disease, or may only partially prevent a disease in a single individual. As such, the terms prevention and prophylaxis may be used interchangeably.
[0059] By the term effective amount of a therapeutic agent is meant a nontoxic but sufficient amount of a beneficial agent to provide the desired effect. The amount of beneficial agent that is effective will vary from subject to subject, depending on the age and general condition of the subject, the particular beneficial agent or agents, and the like. Thus, it is not always possible to specify an exact effective amount. However, an appropriate effective amount in any subject case may be determined by one of ordinary skill in the art using routine experimentation. Also, as used herein, and unless specifically stated otherwise, an effective amount of a beneficial can also refer to an amount covering both therapeutically effective amounts and prophylactically effective amounts.
[0060] An effective amount of a drug necessary to achieve a therapeutic effect may vary according to factors such as the age, sex, and weight of the subject. Dosage regimens can be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation.
[0061] As used herein, a therapeutically effective amount of a therapeutic agent refers to an amount that is effective to achieve a desired therapeutic result, and a prophylactically effective amount of a therapeutic agent refers to an amount that is effective to prevent an unwanted physiological condition. Therapeutically effective and prophylactically effective amounts of a given therapeutic agent will typically vary with respect to factors such as the type and severity of the disorder or disease being treated and the age, gender, and weight of the subject. The term therapeutically effective amount can also refer to an amount of a therapeutic agent, or a rate of delivery of a therapeutic agent (e.g., amount over time), effective to facilitate a desired therapeutic effect. The precise desired therapeutic effect will vary according to the condition to be treated, the tolerance of the subject, the drug and/or drug formulation to be administered (e.g., the potency of the therapeutic agent (drug), the concentration of drug in the formulation, and the like), and a variety of other factors that are appreciated by those of ordinary skill in the art.
[0062] As used herein, the term pharmaceutically acceptable component can refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation of the invention and administered to a subject as described herein without causing any significant undesirable biological effects or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When the term pharmaceutically acceptable is used to refer to an excipient, it is generally implied that the component has met the required standards of toxicological and manufacturing testing or that it is included on the Inactive Ingredient Guide prepared by the U.S. Food and Drug Administration.
[0063] Pharmaceutically acceptable carrier (sometimes referred to as a carrier) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms carrier or pharmaceutically acceptable carrier can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. As used herein, the term carrier encompasses, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations and as described further herein.
[0064] As used herein, pharmaceutically acceptable salt is a derivative of the disclosed compound in which the parent compound is modified by making inorganic and organic, non-toxic, acid or base addition salts thereof. The salts of the present compounds can be synthesized from a parent compound that contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting free acid forms of these compounds with a stoichiometric amount of the appropriate base (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or the like), or by reacting free base forms of these compounds with a stoichiometric amount of the appropriate acid. Such reactions are typically carried out in water or in an organic solvent, or in a mixture of the two. Generally, non-aqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are typical, where practicable. Salts of the present compounds further include solvates of the compounds and of the compound salts.
[0065] Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts include the conventional non-toxic salts and the quaternary ammonium salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. For example, conventional non-toxic acid salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric and the like; and the salts prepared from organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic, mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic, HOOC(CH.sub.2).sub.nCOOH where n is 0-4, and the like, or using a different acid that produces the same counterion. Lists of additional suitable salts may be found, e.g., in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418 (1985).
[0066] Also, as used herein, the term pharmacologically active (or simply active), as in a pharmacologically active derivative or analog, can refer to a derivative or analog (e.g., a salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) having the same type of pharmacological activity as the parent compound and approximately equivalent in degree.
[0067] A control is an alternative subject or sample used in an experiment for comparison purposes. A control can be positive or negative.
[0068] As used herein, by a subject is meant an individual. Thus, the subject can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.), and birds. Subject can also include a mammal, such as a primate or a human. Thus, the subject can be a human or veterinary patient. The term patient refers to a subject under the treatment of a clinician, e.g., physician. Administration of the therapeutic agents can be carried out at dosages and for periods of time effective for treatment of a subject. In some embodiments, the subject is a human.
Abbreviations
[0069] FDA Food and Drug Administration [0070] HBOC Hemoglobin-based oxygen carrier [0071] bHb Bovine hemoglobin [0072] MOF Metal organic framework [0073] ZIF-8 Zeolitic imidazolate framework-8 (C.sub.8H.sub.12N.sub.4Zn) [0074] ZIF-8P-Hb Encapsulated hemoglobin using ZIF-8 precursors [0075] PolybHb Polymerized bovine hemoglobin [0076] DI Deionized water [0077] DLS Dynamic light scattering [0078] Hb Hemoglobin [0079] HF Hollow fiber [0080] hHb Human hemoglobin [0081] Hp Haptoglobin [0082] k.sub.O2.off O.sub.2 dissociation rate constant [0083] MetHb Methemoglobin [0084] MW Molecular weight [0085] n Hill coefficient [0086] NO Nitric oxide [0087] P.sub.50 Partial pressure of O.sub.2 at which 50% of the hemoglobin is saturated with O.sub.2 [0088] PB Phosphate buffer [0089] PBS Phosphate buffered saline [0090] pO.sub.2 Partial pressure of O.sub.2 [0091] PolyHb Polymerized hemoglobin [0092] R-State Relaxed quaternary state [0093] T-State Tense quaternary state [0094] RBC Red blood cell [0095] HPLC-SEC Size exclusion high performance liquid chromatography [0096] TFF Tangential flow filtration [0097] HbV Vesicle encapsulated hemoglobin
Methods
[0098] Provided herein are method for producing a population of matrix-encapsulated protein particles. These methods can comprise: (a) combining a first framework precursor, a second framework precursor, and a protein to form a reactant mixture; (b) incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; and (c) separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration. The resulting matrix-encapsulated protein particles can comprise a protein encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor.
[0099] In some embodiments, step (c) comprises filtering the reactant mixture comprising the matrix-encapsulated protein particles by ultrafiltration against a filtration membrane, thereby forming a retentate fraction comprising matrix-encapsulated protein particles having a molecular weight above a cutoff value and a permeate fraction comprising unencapsulated protein and other impurities having a molecular weight of less than the cutoff value.
[0100] The cutoff value can be between the molecular weight of the protein present in the reaction mixture and the average particle size of the matrix-encapsulated protein particles (i.e., so as to facilitate efficient separation of the matrix-encapsulated protein particles from unencapsulated protein and other impurities remaining in the reactant mixture). In some embodiments, the cutoff value is from 50 kDa to 1000 kDa (e.g., from 150 kDa to 750 kDa, from 250 to 750 kDa, or from 400 kDa to 600 kDa). In certain embodiments, the filtration membrane can be rated for retaining solutes having a molecular weight of greater than 50 kDa, such as greater than 100 kDa, greater than 150 kDa, greater than 250 kDa, greater than 300 kDa, or greater than 500 kDa.
[0101] In certain embodiments, the ultrafiltration can comprise tangential flow filtration or cross-flow filtration.
[0102] The porous framework can comprise, for example, a metal-organic framework (MOF), metal-inorganic framework (MIF), and/or covalent-organic framework (COF). In certain embodiments, the porous framework can comprise a MOF.
[0103] In some embodiments, the first framework precursor can comprise a metal salt and the second framework precursor can comprise a ligand. For example, in some examples, the first framework precursor can comprise a Fe salt, a Co salt, a Cu salt, a Zn salt, or a combination thereof. In some examples, the second precursor can comprise a ligand selected from the group consisting of imidazoles and derivatives such as 2-methylimidazole, 2-ethylimidazole, 4-azabenzimidazole, benzimidazole, nitroimidazole, 2-chloroimidazole, and the like; carboxylic acids and derivatives such as 1,4-benzenedicarboxylic acid, 1,3,5-benzene tricarboxylic acid, imidazole carboxaldehyde, 2-aminobenzimidazolate, the like, or any combination thereof.
[0104] In some embodiments, the first framework precursor and the second framework precursor can be present in the reactant mixture at a molar ratio of from 1:1 to 75:1, such as from 1:1 to 60:1, from 1:1 to 30:1, or from 15:1 to 30:1.
[0105] In certain embodiments, the porous framework can comprise a zeolitic imidazolate framework (ZIF). For example, the porous framework can comprise a zeolitic imidazolate framework such as ZIF-2, ZIF-3, ZIF-4, ZIF-8, ZIF-9, ZIF-10, ZIF-11, ZIF-12, ZIF-14, ZIF-20, ZIF-21, ZIF-23, ZIF-60, ZIF-61, ZIF-62, ZIF-64, ZIF-65, ZIF-67, ZIF-68, ZIF-69, ZIF-70, ZIF-71, ZIF-72, ZIF-73, ZIF-74, ZIF-75, ZIF-76, ZIF-77, ZIF-90, derivatives thereof, and combinations thereof.
[0106] The protein can comprise any suitable protein. For example, the protein can comprise conalbumin, albumin, hemoglobin, haptoglobin, hemopexin, transferrin, methemoglobin, ovalbumin, -chymotrypsinogen A, -chymotrypsin, trypsin, trypsinogen, -lactoglobulin, myoglobin, -lactalbumin, lysozyme, ribonuclease A, or cytochrome c, a recombinant version thereof, or a combination thereof. In some embodiments, the protein can be surface conjugated.
[0107] In some embodiments, the protein can comprise a globular protein. In certain embodiments, the protein can comprise hemoglobin. The hemoglobin can be from a mammalian, invertebrate, or recombinant source. For example, the hemoglobin can comprise human hemoglobin, bovine hemoglobin, or porcine hemoglobin. In some embodiments, the hemoglobin can comprise a polymerized hemoglobin. The polymerized hemoglobin can be in the tense or relaxed quaternary state, or is in between these two quaternary states.
[0108] In some embodiments, the reactant mixture can further comprise an etching agent, a chelating agent, or a combination thereof. For example, the etching agent can comprise hydrofluoric acid (HF), ammonium fluoride (NH.sub.4F), the acid salt of ammonium fluoride
[0109] (NH.sub.4HF.sub.2), sodium hydroxide (NaOH), nitric acid (HNO.sub.3), hydrochloric acid (HCl), hydroiodic acid (HI), hydrobromic acid (HBr), boron trifluoride (BF.sub.3), sulfuric acid (H.sub.2SO.sub.4), acetic acid (CH.sub.3COOH), formic acid (HCOOH), phosphoric acid (H.sub.3PO.sub.4), or any combination thereof. The chelating agent can comprise, for example, ethylenediaminetetraacetic acid (EDTA) or a derivative thereof.
[0110] The resulting population of matrix-encapsulated protein particles can have any suitable size. In some cases, the population of matrix-encapsulated protein particles can comprise microparticles. In other embodiments, the population of matrix-encapsulated protein particles can comprise nanoparticles.
[0111] In certain embodiments, the population of matrix-encapsulated protein particles can have an average particle size, as determined by electron microscopy, of less than 200 nm, such as less than 180 nm, less than 160 nm, less than 140 nm, less than 120 nm, less than 100 nm, or less than 80 nm.
[0112] In other embodiments, the population of matrix-encapsulated particles can have a larger average particle size, such as an average particle size of at least 500 nm, at least 750 nm, at least 1 micron, at least 1.5 microns, at least 2 microns, at least 2.5 microns, at least 5 microns, at least 10 microns, at least 20 microns, at least 30 microns, at least 40 microns, at least 50 microns, or at least 100 microns. These larger particles can be suitable for extracorporeal applications. By way of example, in some examples, these larger particles can include methemoglobin. Such particles can be used to scavenge, for example cyanide, hydrogen sulfide and/or azide, extracorporeally.
[0113] In some embodiments, the population of matrix-encapsulated protein particles can have a PDI of less than 0.100, such as less than 0.095, such as less than 0.090, less than 0.085, less than 0.080, less than 0.075, or less than 0.070.
[0114] In some embodiments, the population of matrix-encapsulated protein particles can have a zeta potential of less than 5 mV, such as of less than 6 mV, less than 7 mV, less than 8 mV, less than 9 mV, less than 10 mV, less than 11 mV, less than 12 mV, less than 13 mV, less than 14 mV, or less than 15 m V.
[0115] In some embodiments, the protein can retain its biological activity following encapsulation. For example, in some embodiments, at least 90% of the of the protein in the population of matrix-encapsulated protein particles retain their biological activity. For example, in the case of methods related to the encapsulation of hemoglobin, in some embodiments, at least 90% of the of the protein in the population of matrix-encapsulated protein particles comprises hemoglobin (and less than 10% of the protein comprises methemoglobin).
[0116] In some embodiments, the methods can encapsulate a protein with relatively high encapsulation efficiency. For example, in some embodiments, the method can encapsulate a protein with an encapsulation efficiency, measured by the fraction of the mass of the protein in the resulting matrix-encapsulated protein over the total mass of protein initially charged in the reactant mixture, of at least 80%, such as at least 82%, at least 84%, at least 86%, at least 88%, at least 90%, at least 92%, at least 94%, or at least 96%.
[0117] In some embodiments, the protein present in the population of matrix-encapsulated protein particles can be modified (e.g., oxidized, reduced, or covalently modified) before and/or after encapsulation. For example, provided are methods for producing a population of matrix-encapsulated protein particles, wherein the protein comprises methemoglobin or polymerized methemoglobin. In some embodiments, these methods can comprise combining a first framework precursor, a second framework precursor, and hemoglobin or polymerized hemoglobin to form a reactant mixture; incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; contacting the population of matrix-encapsulated protein particles with an oxidizing agent under conditions effective to convert the hemoglobin or polymerized hemoglobin to methemoglobin or polymerized methemoglobin; and separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration; wherein the matrix-encapsulated protein particles comprise a protein encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor. In these embodiments, the step of contacting the matrix-encapsulated protein particles with an oxidizing agent can be performed before and/or after the step of separating the matrix-encapsulated protein from the reactant mixture. In other embodiments, these methods can comprise contacting hemoglobin or polymerized hemoglobin with an oxidizing agent under conditions effective to convert the hemoglobin or the polymerized hemoglobin to methemoglobin or polymerized methemoglobin; combining a first framework precursor, a second framework precursor, and the methemoglobin or the polymerized methemoglobin to form a reactant mixture; incubating the reactant mixture under conditions effective to form the population of matrix-encapsulated protein particles; and separating the matrix-encapsulated protein from the reactant mixture using ultrafiltration; wherein the matrix-encapsulated protein particles comprise methemoglobin or polymerized methemoglobin encapsulated in a porous framework formed by reaction of the first framework precursor and the second framework precursor. In some of the above-referenced embodiments, the oxidizing agent can comprise a weak oxidizing agent, such as potassium nitrite or sodium nitrite. In some of the above-referenced embodiments, the polymerized methemoglobin can be in the tense or relaxed quaternary state, or is in between these two quaternary states.
[0118] Also provided are pharmaceutical compositions comprising a population of matrix-encapsulated hemoglobin or polymerized hemoglobin particles prepared using the methods described herein. The compositions can be administered to a subject in need thereof, for example, to treat hypoxia (e.g., hypoxia is at least partially caused by traumatic brain injury or hemorrhagic shock).
[0119] Also provided are pharmaceutical compositions comprising a population of matrix-encapsulated methemoglobin or polymerized methemoglobin particles prepared using the methods described herein. These compositions can be administered to a subject in need thereof, for example, to treat cyanide, hydrogen sulfide, and/or azide poisoning. A population of matrix-encapsulated methemoglobin or polymerized methemoglobin particles can also be employed extracorporeally to treat cyanide, hydrogen sulfide, and/or azide poisoning.
[0120] Reference will now be made in detail to specific aspects of the disclosed materials, compounds, compositions, articles, and methods, examples of which are illustrated in the accompanying Examples and Figures.
EXAMPLES
Example 1: ZIF-8 Metal Organic Framework Encapsulated Hemoglobin
Summary
[0121] Hemoglobin (Hb)-based oxygen carriers (HBOCs) are being developed as artificial red blood cell (RBC) substitutes for use in transfusion medicine. Unfortunately, prior generations of HBOCs were not able to successfully minimize key side effects including vasoconstriction, systemic hypertension and oxidative tissue injury, which is primarily due to the extravasation of cell-free Hb from the vascular space into the tissue space. Therefore, to potentially reduce these side effects, we successfully encapsulated Hb within a zeolitic imidazolate framework (ZIF-8) to form ZIF-8-Hb nanoparticles (ZIF-8P-Hb). Both ZIF-8 and ZIF-8P-Hb nanoparticles were synthesized at a relatively high molar ratio of 2-methylimidazole:zinc, which resulted in a monodisperse nanoparticle size distribution. In addition, the flow conditions for tangential flow filtration facilitated purification of the nanoparticles did not exert a strong effect on the nanoparticle size distribution. ZIF-8P-Hb nanoparticles exhibited high stability, ultrahigh Hb encapsulation efficiency and a monodisperse size distribution. Additionally, ZIF-8P-Hb nanoparticles exhibited a zeta potential of 11.20.9 mV, demonstrating its potentially enhanced biocompatibility in comparison to bare ZIF-8 nanoparticles (40.72.0 mV). More significantly, ZIF-8P-Hb nanoparticles exhibited significantly enhanced hydrothermal stability with negligible release of cell-free Hb. Furthermore, ZIF-8P-Hb displayed a significantly lower haptoglobin binding rate constant compared to cell-free Hb, indicating its potentially slower in vivo clearance in comparison to cell-free Hb. Moreover, we observed a relatively low level of hemolysis when ZIF-8P-Hb nanoparticles were incubated with RBCs (<5%), which demonstrate a suitable safety profile. To further optimize the ZIF-8P-Hb nanoparticle synthesis protocol, various procedural parameters were systematically investigated to evaluate their impact on the size distribution of ZIF-8 and ZIF-8P-Hb nanoparticles. Taken together, this work provides a comprehensive approach for synthesizing a monodisperse HBOC as a potential artificial RBC substitute.
Introduction
[0122] Metal organic frameworks (MOFs) are crystalline materials synthesized by the coordination of metal ions and organic linkers. Zeolitic imidazolate frameworks (ZIFs) are a subclass of MOF, which exhibits outstanding solvothermal stability due to their unique structure compared to other MOFs. ZIF-8, the prototypical ZIF, preserves its crystallinity and zeolite-like porosity when exposed to boiling water, organic solvents, and several biological buffers. Very recently, ZIF-8 has emerged as a promising material in biological sensing, and pharmaceutical applications.
[0123] Recently, ZIF-8 encapsulated Hb (ZIF-8@Hb) nanoparticles have been prepared, which improved survival in a murine hemorrhagic shock model compared to that of transfused cell-free Hb. Despite relatively high Hb encapsulation efficiency, the absolute amount of Hb loaded into ZIF-8 nanoparticles was quite low likely due to the interference of Hb on ZIF-8 nanoparticle nucleation. Additionally, the final ZIF-8@Hb nanoparticle suspension exhibited a dark green color, which qualitatively indicated excessive formation of methemoglobin (metHb). MetHb is the oxidized form of ferrous Hb, which cannot bind O.sub.2.
[0124] In this Example, bovine Hb (bHb) was successfully encapsulated using ZIF-8 precursors into ZIF-8P-Hb nanoparticles and exhibited low oxygen affinity. Bovine Hb encapsulation was performed in the presence of a 30:1 molar ratio of 2-methylimidazole (Hmim) to zinc to reduce ZIF-8P-Hb nanoparticle polydispersity and size by facilitating the nanoparticle nucleation process. ZIF-8P-Hb nanoparticles were then purified via tangential flow filtration (TFF) to remove unreacted ligands and residual cell-free Hb. TFF was found to have more flexibility and scalability compared to conventional centrifugation method since centrifuges have a finite limit on the size of the containers, hindering the overall feasibility of large scale manufacturing via centrifugation. ZIF-8P-Hb nanoparticles were able to maintain their oxygen binding/release capabilities. Moreover, protection of encapsulated Hb against recognition by haptoglobin was found to be substantial given that the haptoglobin binding rate constant was significantly lower for ZIF-8P-Hb nanoparticles compared to that of cell-free bHb. Furthermore, the significantly reduced zeta potential of ZIF-8P-Hb nanoparticles compared to bare ZIF-8 nanoparticles suggests potential biocompatibility. Finally, the low level of hemolysis (<5%) induced by ZIF-8P-Hb nanoparticles demonstrate suitable hemocompatibility for its potential use as an RBC substitute in treating hemorrhagic shock. Further optimization was performed by evaluating the effect of multiple procedural parameters on the size distribution and polydispersity of ZIF-8 and ZIF-8P-Hb nanoparticles.
Methods
Bovine Hb Purification
[0125] Bovine Hb (bHb) was purified via tangential flow filtration (TFF) as described previously in the literature. Briefly, Hb was purified via a two-stage TFF system with hollow fiber cartridges with MWCOs of 500 and 50 kDa (Repligen Corporation, Rancho Dominguez, CA). The purified bHb was concentrated to >200 mg/mL and stored at 80 C. for future use.
Synthesis and Purification of ZIF-8-Hb Nanoparticles
[0126]
Synthesis Parameter Optimization
[0127] To optimize the protocol for synthesizing ZIF-8 and ZIF-8P-Hb nanoparticles, the effect of various synthesis parameters on the biophysical properties of ZIF-8/ZIF-8P-Hb nanoparticles were evaluated. More specifically, the initial concentration of bHb, the concentration of EDTA, the flow rate of the peristaltic pump during the diafiltration process, the concentration of zinc nitrate, the molar ratio of Hmim:Zn, and different addition methods for introduction of Hmim and zinc nitrate hexahydrate into the reaction vessel were varied in this study
Hydrodynamic Diameter
[0128] The hydrodynamic diameter of bHb, ZIF-8 and ZIF-8P-Hb nanoparticles were measured using a BI-200SM goniometer (Brookhaven Instruments Corp., Holtsville, NY) at an angle of 90 and wavelength of 637 nm. Protein samples were diluted to 0.5-1 mg/mL concentration in DI water. The hydrodynamic diameter was calculated via the instrument software.
Morphology and Crystalline Structure
[0129] The morphology of ZIF-8 and ZIF-8P-Hb nanoparticles was studied with a Thermo Glacios cryo-electron microscopy (Thermo Fisher Scientific). Protein samples were diluted to 0.5-1 mg/mL concentration in DI water. XRD patterns were recorded on a Bruker D8 Advance diffractometer (AXS, Bruker, Germany) with Cu target from 5 C. to 55 C.
Oxygen (O.SUB.2.) Equilibrium Curves
[0130] O.sub.2 equilibrium curves (OECs) for bHb and ZIF-8P-Hb nanoparticles were measured using a Hemox Analyzer (TCS Scientific Corp., New Hope, PA) at 37.00.1 C. in phosphate buffered saline (PBS, 0.1 M, pH 7.4). To quantify the oxygen binding affinity (P.sub.50) and cooperativity coefficient (n), the OEC was fit to the Hill equation to regress the parameters as described in the literature.
Rapid Deoxygenation Kinetics
[0131] Initially, bHb and ZIF-8P-Hb nanoparticles were diluted to 12.5 M (heme basis) in PBS (0.1 M, pH 7.4). Deoxygenated buffer was prepared by adding 1.5 mg/mL of sodium dithionite to PBS bubbled under N.sub.2 for 30 minutes. Deoxygenated buffer and either oxygenated bHb orZIF-8P-Hb nanoparticles were mixed rapidly in a microvolume stopped-flow spectrophotometer (Applied Photophysics Ltd., Surrey, United Kingdom) and the absorbance was monitored at 437.5 nm. An exponential decay function was fit to the data and the rate constant for O.sub.2 dissociation (k.sub.off.O2) was regressed for each sample.
Haptoglobin Binding Kinetics
[0132] The kinetics of haptoglobin (Hp) binding to bHb or ZIF-8P-Hb nanoparticles was measured in PBS (0.1 M, pH 7.4) as described in the literature. The reaction between Hp and bHb or ZIF-8P-Hb nanoparticles was monitored by stopped flow fluorescence spectrometry (.sub.excitation=285 nm, .sub.emission=310 nm) as previously described in the literature. The pseudo first order Hp binding rate constant was calculated by fitting the fluorescence intensity to a mono-exponential equation. The pseudo first-order rate constants as a function of [Hb] was then used to determine the bimolecular rate constant via linear regression.
Hb Release from ZIF-8P-Hb Nanoparticles
[0133] Hb release from ZIF-8P-Hb nanoparticles was studied as a function of the bHb release rate as described in the literature. Briefly, bHb release from ZIF-8P-Hb nanoparticles was measured over 14 days at 4 C. in PBS (0.1 M, pH 7.4). The permeate was collected by filtering the stored nanoparticle solution through a 500 kDa TFF membrane. The concentration of bHb from the resulting permeate was measured using UV-visible spectroscopy.
Hemocompatibility of ZIF-8P-Hb Nanoparticles
[0134] Expired human RBCs and plasma units were generously donated by Transfusion Services, Wexner Medical Center, The Ohio State University (Columbus, OH). The hemolysis assay was performed as described in the literature. Briefly, 1 mL of human RBCs was mixed with 1 mL of ZIF-8P-Hb nanoparticles suspended in 0.9% saline solution. The mixtures were incubated at 37 C. in a water bath incubator for 30 minutes and then centrifuged to collect the supernatant. The bHb concentration of the supernatant was measured with UV-visible spectroscopy. Hemolysis was determined by the ratio of supernatant concentration of bHb in ZIF-8P-Hb nanoparticle/RBC mixture to the total Hb concentration derived from the RBCs.
Total bHb and Methemoglobin (MetHb) Levels
[0135] Total bHb and metHb concentrations were determined using the cyanmethemoglobin method. To accurately measure the bHb concentration and reduce the effect of nanoparticle scattering, the cyanmethemoglobin method was slightly modified by adding EDTA (0.5M) to fully dissolve the ZIF-8P-Hb nanoparticle crystalline structure prior to the addition of cyanide. Spectrophotometric absorbance measurements were obtained using a HP 8452A diode array spectrophotometer (Olis, Bogart, GA).
bHb Encapsulation Efficiency and Loading
[0136] The encapsulation efficiency (EE %) and loading of bHb inside ZIF-8P-Hb nanoparticles was calculated based on the total mass fraction of encapsulated bHb present in the retentate solution as shown in Eqs 1 and 2, respectively, where Hb.sub.init corresponds to the mass of bHb in the permeate solution at the end of the TFF washing process and Hb.sub.init corresponds to the initial mass of bHb.
Statistical Analysis
[0137] In this study, all statistical analysis was performed using a t-test, and a p value of <0.05 was considered significant.
Results and Discussion
[0138] Table 1 lists the biophysical properties of ZIF-8 and ZIF-8P-Hb nanoparticles in comparison to other types of HBOCs. The effect of bHb encapsulation using ZIF-8 precursors on the biophysical properties of ZIF-8P-Hb nanoparticles was studied by comparing the hydrodynamic diameter, zeta potential, oxygen equilibria, oxygen offloading rate constant, and Hp binding rate constant to native bHb and other HBOCs from the literature.
TABLE-US-00001 TABLE 1 Biophysical properties of bHb, ZIF-8 and ZIF-8P-Hb nanoparticles, and other types of HBOCs described in the literature including R-state PolybHb 30:1 (R30), T-state PolybHb 35:1 (T35), and vesicle encapsulated Hb (HbV). R30 and T35 represent typical polymerized Hbs synthesized via glutaraldehyde cross-linking under fully oxygenated (relaxed quaternary state [R]) and deoxygenated (tense quaternary state [T]) conditions at glutaraldehyde:bHb molar ratios of 30:1 and 35:1. HbV represent a phospholipid bilayer membrane (liposome) encapsulating an aqueous core of concentrated Hb molecules. The biophysical properties of RBCs was also included in the table since it is the natural oxygen carrier in most organisms with a circulatory system. Parameter bHb ZIF-8 ZIF-8P-Hb R30 T35 HbV RBCs P.sub.50 (mm Hg) 26.3 0.5 11.8 0.6 1.2 0.4 38.9 2.5 32 25.6 Cooperativity (n) 2.50 0.10 1.99 0.08 1.0 0.2 1.0 0.1 2.5 2.8 MW (kDa) 65 1,131 97 1,194 106 D.sub.eff (nm) 6.9 0.3 90.3 11.8 106.0 9.7 62.4 12.5 64.1 9.3 251 87 7-8 10.sup.3 PDI 0.1 0.065 0.006 0.085 0.008 0.24 0.02 0.19 0.02 Zeta potential (mV) 21.2 2.5 40.7 2.0 11.2 0.9 11.1 0.6 22.5 1.3 18 12.5 k.sub.off, O2 (s.sup.1) 45.86 1.00 19.57 1.27 15.12 1.60 35.13 9.90 9.4 5.1 k.sub.Hb-Hp (M.sup.1s.sup.1) 0.1491 0.0405 0.0076 0.0228 MetHb level (%) 1.0 0.3 4.1 0.4 3.7 1.1 3.2 1.2 <10 <1
Crystalline Structure and Morphology
[0139] In
Size and Zeta Potential
[0140] It was observed that ZIF-8 nanoparticles synthesized in this study possessed a hydrodynamic diameter of 90.311.8 nm with a relatively low polydispersity (PDI) of 0.065 #: 0.006 as shown in Table 1. After bHb encapsulation, ZIF-8P-Hb nanoparticles exhibited a hydrodynamic diameter of 106.09.68 nm with narrow size distribution (PDI=0.0850.008). In comparison to ZIF-8@Hb nanoparticles (diameter ranging from 164.5-365.2 nm) synthesized in the literature, the size of ZIF-8P-Hb nanoparticles (diameter 100 nm) synthesized in this current study is more advantageous, since nanoparticles with larger particle sizes (>300 nm) are more prone to uptake by the reticuloendothelial system (RES) and being trapped in the hepatic sinusoids.
[0141] Surface charge changes were assessed by measuring the zeta potential before and after bHb encapsulation inside the ZIF-8 nanoparticle (Table 1). In
Procedural Meta-Data Analysis
[0142]
[0143]
Oxygen Affinity and Offloading Kinetics
[0144]
[0145] The k.sub.off.O2 of ZIF-8P-Hb nanoparticles (19.57+1.27 s.sup.1) was significantly higher compared to HbV (9.4 s.sup.1) and RBCs (5.1 s.sup.1), which could be due to the porous structure of ZIF-8P-Hb nanoparticles which facilitated faster O.sub.2 diffusion through the particle core (
Haptoglobin Binding Kinetics.
[0146]
bHb Encapsulation Efficiency and bHb Leakage
[0147] In general, cell-free bHb not removed by diafiltration would be included in the quantity of particle entrapped Hb as shown in Eq 1. In this study, ultrahigh bHb encapsulation efficiency (EE %=88.23.5%) of ZIF-8P-Hb nanoparticles was achieved, indicating favorable oxygen carrying capacity. In comparison to the EE % of HbV prepared via both the conventional extrusion method (20%) and the kneading method (74.2%), the encapsulation of bHb using ZIF-8 precursors yielded a significantly higher EE % likely because the nucleation and encapsulation process were both rapid due to the high molar ratio of Hmim:Zn (30:1). ZIF-8P-Hb nanoparticles also exhibited higher EE % and Hb loading (0.81 mg Hb/1 mg ZIF-8P-Hb) than that of ZIF-8@Hb nanoparticles (EE %=82.1%, Hb loading=0.03-0.24 mg Hb/1 mg ZIF-8@Hb) as described in the literature. The Hb loading of ZIF-8@Hb nanoparticles was estimated by assuming 100% yield of the ZIF-8@Hb nanoparticles based on the mass of the zinc source.
[0148] To measure the long-term stability of ZIF-8P-Hb nanoparticles, bHb leakage was evaluated at 4 C. for 14 days. In
Hemocompatibility
[0149] Hemolysis is characterized by the rupture of RBCs and the release of cell-free Hb. Cell-free Hb can elicit renal failure, and tissue oxidative injury. Thus, it is important to measure the hemolytic activity of ZIF-8P-Hb nanoparticles as a guide to their potential biocompatibility with blood. In this study, hemolysis was assayed following 30 minutes incubation at 37 C. with RBCs. In
Conclusion
[0150] In general, the size of ZIF-8P-Hb nanoparticles was primarily controlled by Hmim:Zn molar ratio, flow rate during TFF processing, concentration of EDTA, and concentration of zinc nitrate. It was found that adding Hmim directly into the reaction vessel regulated particle size, which also affected the crystalline structure of the particle. Furthermore, we demonstrated that the high molar ratio of Hmim:Zn could be used to better control nucleation of ZIF-8P-Hb nanoparticles. The monodisperse size distribution was a result of the rapid nucleation rate facilitated by the relatively high molar ratio of Hmim:zinc, and TFF operated at relatively low flow rate did not exert a strong impact on the size distribution. Therefore, we established a scalable purification platform to manufacture ZIF-8 and ZIF-8P-Hb nanoparticles via TFF. The optimized synthesis protocol yielded relatively low batch-to-batch variance with respect to most biophysical properties including hydrodynamic diameter, zeta potential, oxygen equilibria, and oxygen offloading rate constant. The higher bHb loading and bHb encapsulation efficiency of ZIF-8P-Hb nanoparticles compared to prior attempts in the literature is critical in order to potentially use these materials to treat hemorrhagic shock. Future analysis of these materials will need to evaluate ZIF-8P-Hb nanoparticle efficacy and safety in vivo.
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Influence of Shear Forces on the Aggregation and Sedimentation Behavior of Cerium Dioxide (CeO2) Nanoparticles under Different Hydrochemical Conditions. J. Nanoparticle Res. 2016, 18 (7). https://doi.org/10.1007/s11051-016-3501-3. [0190] (40) Jian, M.; Liu, B.; Liu, R.; Qu, J.; Wang, H.; Zhang, X. Water-Based Synthesis of Zeolitic Imidazolate Framework-8 with High Morphology Level at Room Temperature. RSC Adv. 2015, 5 (60), 48433-48441. https://doi.org/10.1039/c5ra04033 g. [0191] (41) Cravillon, J.; Schrder, C. A.; Nayuk, R.; Gummel, J.; Huber, K., Wiebcke, M. Fast Nucleation and Growth of ZIF-8 Nanocrystals Monitored by Time-Resolved in Situ Small-Angle and Wide-Angle X-Ray Scattering. Angew. ChemieInt. Ed. 2011, 50 (35), 8067-8071. https://doi.org/10.1002/anie.201102071. [0192] (42) Kure, T.; Sakai, H. Preparation of Artificial Red Blood Cells (Hemoglobin Vesicles) Using the Rotation-Revolution Mixer for High Encapsulation Efficiency. ACS Biomater. Sci. 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Example 2. ZIF-8 Metal Organic Framework Nanoparticle Loaded with Tense Quaternary State Polymerized Bovine Hemoglobin: Potential Red Blood Cell Substitute with Antioxidant Properties
Overview
[0193] Due to several limitations associated with blood transfusion, such as the relatively short shelf life of stored blood, low risks of acute immune hemolytic reactions and graft-versus-host disease, many strategies have been developed to synthesize hemoglobin-based oxygen carriers (HBOCs) as universal red blood cell (RBC) substitutes. Recently, zeolite imidazole framework-8 (ZIF-8), a metal-organic framework, has attracted considerable attention as a protective scaffold for encapsulation of hemoglobin (Hb). Despite the exceptional thermal and chemical stability of ZIF-8, the major impediments to implementing ZIF-8 for Hb encapsulation are the structural distortions associated with loading large quantities of Hb in the scaffold as the Hb molecule has a larger hydrodynamic diameter than the pore size of ZIF-8. Therefore, to reduce the structural distortion caused by Hb encapsulation, we established and optimized a continuous-injection method to synthesize nanoparticle (NP) encapsulated polymerized bovine Hb (PolybHb) using ZIF-8 precursors (ZIF-8P-PolybHb NPs). The synthesis method was further modified by adding EDTA as a chelation-assisted etching agent, which reduced the ZIF-8P-PolybHb NP size to <300 nm. ZIF-8P-PolybHb NPs exhibited low oxygen affinity (36.4+3.2 mm Hg) compared to unmodified bovine Hb but was similar in magnitude to unencapsulated PolybHb. The use of the chemical cross-linker glutaraldehyde to polymerize bovine Hb resulted in the low Hill coefficient of PolybHb, indicating loss of Hb's oxygen binding cooperativity, which could be a limitation when using PolybHb as an oxygen carrier for encapsulation inside the ZIF-8 matrix. ZIF-8P-PolybHb NPs exhibited slower oxygen offloading kinetics compared to unencapsulated PolybHb, demonstrating successful encapsulation of PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant properties when exposed to H.sub.2O.sub.2. Incorporation of PolybHb into the ZIF-8 scaffold resulted in improved cytotoxicity in human umbilical vein endothelial cells compared to unloaded ZIF-8 NPs and ZIF-8 NPs loaded with bovine Hb. We envisage that such a monodisperse and biocompatible HBOC with low oxygen affinity and antioxidant properties may broaden its use as an RBC substitute.
Introduction
[0194] Red blood cell (RBC) transfusion is a routine medical procedure to treat patients suffering from substantial surgical or traumatic blood loss; however, the relatively short ex vivo shelf-life of blood (42 days) and low probability risks of contracting unknown blood borne pathogens are the major concerns associated with RBC transfusion. To address these concerns, hemoglobin-based oxygen carriers (HBOCs) have been developed and tested in clinical trials as RBC substitutes over the last several decades. Unfortunately, none of these commercial HBOCs received FDA approval for clinical use in the U.S. due to severe adverse events observed during Phase III clinical trials. For instance, the presence of extracellular hemoglobin (Hb) and low molecular weight species (<300 kDa) in previous generations of HBOCs elicited vasoconstriction, systemic hypertension, myocardial infarction, acute renal damage, and heart lesions. This provides the rationale for the development of safer HBOCs.
[0195] Metal organic frameworks (MOFs) recently emerged as an excellent protective material to encapsulate biomolecules, cells, and DNA due to their superior solvothermal stability. ZIF-8, as a subclass of MOFs, is comprised of tetrahedral Zn.sup.2+ coordinated to 2-methlyimidazolate (Hmim) arranged in porous structures. Its remarkably high surface area and large pore size could potentially favor oxygen (O.sub.2) storage and delivery. ZIF-8-ncapsulated Hb (ZIF-8@Hb) can possess extended circulation times in a murine model when compared to cell-free Hb. Additionally, ZIF-8's high tolerance towards basic environments, oxidation and high temperature can also be found to be present in ZIF-8@Hb. Hb can be encapsulated inside nanoparticles (NPs) using a porous coordination network (PCN)-333 (Al) of Al.sup.3+ connected by the organic linker triazine-2,4,6-triyl-tribenzoic acid. The resultant MOF-NPs encapsulating Hb can bind and release O.sub.2.
[0196] Overall, MOF-encapsulated Hbs prepared to date exhibits relatively low Hb loading (<1 mg/mL), which limits their use as an RBC substitute. To address that issue, we developed ZIF-8 encapsulated bovine Hb NPs (ZIF-8P-Hb NPs) via synthesis at an ultrahigh molar ratio of Hmim:Hb, which facilitates the nucleation reaction. The resulting ZIF-8P-Hb NPs exhibited high structural stability, ultrahigh Hb encapsulation efficiency and loading capacity, and a monodisperse size distribution. However, we observed an increased O.sub.2 affinity for ZIF-8P-Hb NPs when compared to cell-free Hb. In addition, HBOCs with relatively low O.sub.2 affinity were found to be more favorable at delivering O.sub.2 to surrounding tissues. Therefore, in this Example, we describe the encapsulation of tense quaternary state (T-state) polymerized bovine Hb (T-state PolybHb) into ZIF-8 precursors with the aim of designing a ZIF-8 HBOC with low O.sub.2 affinity. T-state PolybHb was able to retain its low O.sub.2 affinity by cross-linking bHb in the T-state with the chemical cross-linker glutaraldehyde. We also established and optimized a continuous-injection method coupled with a chelation-assisted chemical etching process to synthesize ZIF-8P-PolybHb NPs. ZIF-8P-PolybHb NPs exhibited a diameter of 170.917.4 nm and relatively narrow size distribution (PDI=0.0420.011). ZIF-8P-PolybHb NPs had a significantly lower O.sub.2 affinity (36.4+3.2 mm Hg) in comparison to ZIF-8P-Hb NPs (P.sub.50=11.8 mm Hg). Additionally. ZIF-8P-PolybHb NPs exhibited a more negative zeta potential than ZIF-8P-Hb NPs and prior generations of MOF-Hb NPs, demonstrating potentially improved biocompatibility. Favorable antioxidant (H.sub.2O.sub.2) properties and hemocompatibility (<5% hemolysis of RBCs) were also observed for ZIF-8P-PolybHb NPs. Therefore, encapsulation of T-state PolybHb using ZIF-8 precursors should not only be able to facilitate tissue O.sub.2 offloading, but also enhance biocompatibility.
Materials and Methods
Bovine Hb Purification.
[0197] Bovine Hb (bHb) was purified from sodium citrate anticoagulated whole blood (Quad Five, Ryegate, MT) via TFF. Two HF cartridges with MWCOs of 500 and 50 kDa (Repligen Corporation, Rancho Dominguez, CA) were used to purify and concentrate bHb. The final product was stored at >200 mg/mL in the freezer (80 C.) for future use.
bHb Polymerization and PolybHb Purification.
[0198] Glutaraldehyde was used as the chemical cross-linker to synthesize tense quaternary state (T-state) polymerized bHb (PolybHb).
[0199] After PolybHb synthesis, clarification, purification, and concentration of PolybHb was performed via a two-stage TFF process. Specifically, a 500 kDa hollow fiber (HF) module was used to clarify the PolybHb by removing the high MW species (>500 kDa). A 100 kDa TFF module was then used to retain and buffer exchange the low MW PolybHb (>100 kDa but <500 kDa) into PBS (0.1 M, pH 7.4) for subsequent encapsulation by ZIF-8 precursors.
Synthesis and Purification of ZIF-8-PolybHb NPs.
[0200]
Electron Microscopy and X-Ray Diffraction Analysis.
[0201] The morphology of ZIF-8, ZIF-8P-Hb and ZIF-8P-PolybHb NPs was imaged via a FEI Tecnai G2 Biotwin transmission-electron microscope (Thermo Fisher Scientific). Protein samples were diluted to 0.5-1 mg/mL in DI water. XRD spectra were recorded on a Bruker D8 Advance diffractometer (AXS, Bruker, Germany) with Cu target from 5 to 45.
Hydrodynamic Diameter.
[0202] The hydrodynamic diameter of ZIF-8P-PolybHb NPs were measured via dynamic light scattering (DLS) analysis using a BI-200SM goniometer (Brookhaven Instruments Corp., Holtsville, NY) at an angle of 90 and wavelength of 637 nm. Protein samples were diluted to 0.5-1 mg/mL in DI water. The hydrodynamic diameter was calculated via the instrument software.
Zeta () Potential.
[0203] The potential of bHb, PolybHb, ZIF-8P-Hb and ZIF-8P-PolybHb was measured using a Brookhaven Instruments ZetaPals instrument (Holtsville, NY) at room temperature. All samples were diluted to 1 mg/ml in DI water.
Electron Microscopy and X-ray Diffraction Analysis.
[0204] The morphology of ZIF-8, ZIF-8P-Hb and ZIF-8P-PolybHb NPs was imaged via a FEI Tecnai G2 Biotwin transmission-electron microscope (Thermo Fisher Scientific). Protein samples were diluted to 0.5-1 mg/mL in DI water. XRD spectra were recorded on a Bruker D8 Advance diffractometer (AXS, Bruker, Germany) with Cu target from 5 to 45
O.SUB.2 .Equilibrium Analysis.
[0205] To study the O.sub.2 equilibrium properties at 37 C., the O.sub.2 equilibrium curves (OECs) for bHb, PolybHb, ZIF-8P-Hb and ZIF-8P-PolybHb were measured using a Hemox Analyzer (TCS Scientific Corp., New Hope, PA) in PBS (0.1 M, pH 7.4). The OEC was fit to the Hill equation to regress the O.sub.2 binding affinity (P.sub.50, partial pressure of O.sub.2 (pO.sub.2) at which the Hb is half saturated with 02) and Hill coefficient (n).
Rapid O.SUB.2 .Offloading Kinetics.
[0206] The (2 offloading kinetics of bHb, PolybHb, ZIF-8P-Hb NPs and ZIF-8P-PolybHb NPs was studied via rapidly mixing Na.sub.2S.sub.2O.sub.4 solution (1.5 mg/mL) with protein samples (12.5 M, heme basis) in a microvolume stopped-flow spectrophotometer (Applied Photophysics Ltd., Surrey, United Kingdom). The resultant absorbance change was monitored at 437.5 nm and fit to a mono-exponential function to regress the rate constant for O.sub.2 offloading (k.sub.off,O2).
Haptoglobin Binding Kinetics.
[0207] The binding kinetics between human haptoglobin (Hp) and bHb or PolybHb or ZIF-8P-PolybHb NPs was monitored via stopped flow fluorescence spectrometry (.sub.excitation=285 nm, .sub.emission=310 nm) in PBS (0.1 M, pH 7.4). The pseudo first order Hp binding rate constant was calculated by fitting the fluorescence intensity to a mono-exponential equation. The pseudo first order rate constants were then linearly regressed as a function of Hb concentration to obtain the bimolecular rate constant.
Auto-Oxidation Kinetics.
[0208] All samples including bHb, PolybHb, and ZIF-8P-PolybHb NPs were first diluted to 0.31 mM (heme basis) in PBS (0.1 M, pH 7.4) and submerged in water bath at 37 C. for 24 hours. The spectra (300-700 nm) of each sample were collected via UV-visible spectrometry every 60 mins. The metHb concentration was measured using the cyanmethemoglobin method. The auto-oxidation rate constant was calculated by fitting the normalized Hb concentration (ln([Hb.sup.2+]/[Hb.sup.2+].sub.0; ([Hb.sup.2+], the concentration of oxyHb) to a linear function.
Hb Release from ZIF-8P-Hb NPs.
[0209] To study the hydrothermal stability of ZIF-8P-PolybHb NPs, PolybHb release from ZIF-8P-PolybHb NPs was recorded over 7 days at 37 C. in PBS (0.1 M, pH 7.4). The mass of PolybHb in the pooled permeate was calculated by measuring the PolybHb concentration using UV-visible spectrometry and measuring the total permeate volume after filtering the ZIF-8P-PolybHb NP solution through a 500 kDa mPES TFF cartridge.
Hemocompatibility of ZIF-8P-PolybHb NPs.
[0210] Expired human RBCs and plasma units were generously donated by Transfusion Services, Wexner Medical Center, The Ohio State University (Columbus, OH). The hemolysis assay was performed using know methods. Briefly, 1 mL of human RBCs was mixed with 1 mL of ZIF-8P-PolybHb solution suspended in 0.9% saline solution. The mixtures were incubated at 37 C. in a water bath incubator for 30 minutes and then centrifuged to collect the supernatant. The Hb concentration of the supernatant was measured with UV-visible spectrometry. Hemolysis was determined by the ratio of the mass of Hb in the supernatant of the ZIF-8P-PolybHb/RBC mixture to the total mass of Hb derived from the RBCs.
Total bHb and Methemoglobin (MetHb) Levels.
[0211] Total bHb and metHb concentrations were determined using the cyanmethemoglobin method. To accurately measure the bHb concentration and reduce the effect of NP scattering, the cyanmethemoglobin method was slightly modified by adding EDTA (80 mM) to fully dissolve the ZIF-8P-PolybHb NP crystalline structure prior to the addition of cyanide. Spectrophotometric absorbance measurements were obtained using a HP 8452A diode array spectrophotometer (Olis, Bogart, GA).
H.sub.2O.sub.2 Oxidation Kinetics.
[0212] To investigate the antioxidant properties of ZIF-8P-PolybHb NPs against H.sub.2O.sub.2, bHb, PolybHb, ZIF-8P-Hb and ZIF-8P-PolybHb NPs were exposed to an excessive amount of H.sub.2O.sub.2 (100 mM) at different concentrations of total Hb ([Hb]: 0.125, 0.25, 0.5 mg/mL). The change in fractional composition of hemichrome, oxyHb, and metHb was evaluated through spectral deconvolution via an open-source Python package Alchromy (www.alchromy.com).
PolybHb Encapsulation Efficiency.
[0213] The encapsulation efficiency (EE %) of bHb inside ZIF-8P-PolybHb NPs was calculated using the equation below.
where Hb.sub.per corresponds to the mass of PolybHb in the permeate solution at the end of the TFF washing process and Hb.sub.init corresponds to the initial mass of PolybHb.
Cytotoxicity.
[0214] The cytotoxicity of ZIF-8-PolybHb NPs was assessed with an MTS assay using human umbilical vein endothelial cells (HUVECs). Prior to testing, cells were grown in human large vessel endothelial cell basal media supplemented with large vessel endothelial supplement and 1% penicillin-streptomycin until 80-90% confluence. Passages 2-4 were used for testing. Cells were seeded at 110.sup.5/mL in a 96-well plate and left to adhere for 24 hours before application of PolybHb, unloaded ZIF-8 NPs, ZIF-8-bHb NPs, and ZIF-8-PolybHb NPs at varying concentrations (0-500 g/mL). Cells were incubated with treatment groups for 24 hours before staining with MTS reagent for 3 hours. Media and DMSO were used as a positive and negative control, respectively. The optical density at 490 nm was measured with a Varioskan Lux multimode microplate reader using SkanIT software (Thermo Fisher Scientific, Waltham, MA).
Statistical Analysis.
[0215] In this Example, all statistical analysis was performed using a t-test, and a p value of <0.05 was considered significant.
TABLE-US-00002 TABLE 2 Summary of the biophysical properties of ZIF-8, ZIF-8P-Hb, and ZIF-8P-PolybHb in comparison to bHb, PolybHb, HbV, and RBCs. The effect of PolybHb encapsulation using ZIF-8 precursors on the biophysical properties of ZIF-8P-PolybHb NPs was studied by comparing the hydrodynamic diameter, zeta potential, oxygen equilibria, oxygen offloading rate constant, and Hp binding rate constant to native bHb and other HBOCs from the literature. ZIF-8P- Parameter bHb ZIF-8 ZIF-8P-Hb PolybHb PolybHb HbV RBCs P.sub.50 (mm Hg) 26.3 0.5 11.8 0.6* 36.4 3.2* 39.6 2.3* 32 25.6 Cooperativity (n) 2.50 0.10 1.99 0.08* 1.07 0.21* 1.08 0.03* 2.5 2.8 Average MW (kDa) 65 216 D.sub.eff (nm) 6.9 0.3 90.3 11.8* 106.0 9.7* 170.9 17.4* 11.2 1.5* 251 87 7-8 10.sup.3 PDI 0.1 0.065 0.006 0.085 0.008 0.042 0.011 0.3 Zeta potential (mV) 5.8 0.7 40.7 2.0* 11.2 0.9* 15.0 2.3* 17.1 1.4* 18 12.5 k.sub.off, O2 (s.sup.1) 45.86 1.00 19.57 1.27* 16.96 2.62* 39.96 3.45 9.4 5.1 k.sub.Hb-Hp (M.sup.1s.sup.1) 0.1491 0.0405 0.0064 0.0185 k.sub.0X (h.sup.1) 0.0123 0.0019 0.0066 0.0007 0.0068 0.0009 0.0068 0.0005 MetHb level (%) 1.0 0.3 4.1 0.4* 8.30 0.6* 5.5 0.2* <10 <1 (*denotes statistical significance (p < 0.05) compared to bHb.
Results and Discussion
Crystalline Structure and Morphology.
[0216]
Size Distribution.
[0217] According to TEM analysis, ZIF-8 NPs, ZIF-8P-Hb NPs, and ZIF-8P-PolybHb NPs were found to have an average diameter of 80.53 nm, 102.33 nm, and 145.25 nm, which are close to the DLS results (
Zeta Potential.
[0218] To study the effect of PolybHb encapsulation on the surface charge of ZIF-8P-PolybHb NPs, we measured the zeta potential of ZIF-8P-PolybHb NPs in comparison to ZIF-8 NPs, ZIF-8P-Hb NPs, and PolybHb. In
Oxygen Affinity and Offloading Kinetics.
[0219] The O.sub.2 affinity of PolybHb and ZIF-8P-PolybHb NPs was assessed by measuring the O.sub.2 equilibrium curves (OECs), which were then fit to the Hill equation to regress the P.sub.50 (partial pressure of O.sub.2 at which Hb is half saturated with O.sub.2) and n (Hill coefficient). In
[0220] In
Haptoglobin Binding Kinetics.
[0221] The pseudo first order Hp binding kinetics of bHb, PolybHb, ZIF-8P-Hb NPs, and ZIF-8P-PolybHb NPs are shown in
Auto-Oxidation Kinetics.
[0222] The auto-oxidation kinetics of the materials under physiological conditions was monitored by measuring the metHb level of bHb, PolybHb, and ZIF-8P-PolybHb NPs in PBS (0.1 M, pH 7.4) at 37 C. over a 24-hr period. The metHb level was assessed via the cyanmethemoglobin method. In
H.sub.2O.sub.2 Oxidation Kinetics.
[0223] Reactive oxygen species (ROS) including singlet oxygen (.sup.1O.sub.2), hydroxyl radical (.sup.OH), peroxyl radical (.sup.OOR), superoxide (.sup.O.sub.2.sup.), and hydrogen peroxide (H.sub.2O.sub.2) are generated from O.sub.2 mainly inside the mitochondria during cellular metabolism. Endogenous H.sub.2O.sub.2 is known to cause oxidative damage of cellular proteins e.g., Hb, which would be even more extensive for cell-free Hb due to the lack of catalase. To investigate the antioxidant properties of ZIF-8P-PolybHb NPs against H.sub.2O.sub.2, bHb, PolybHb, and ZIF-8P-PolybHb NPs were exposed to H.sub.2O.sub.2 at different concentrations ([Hb], 0.125, 0.25, 0.5 mg/mL).
PolybHb Encapsulation Efficiency (EE %) and Hb Loading.
[0224] The overall Hb encapsulation efficiency (EE %) of ZIF-8P-PolybHb NPs was calculated based on the total mass of encapsulated PolybHb and the initial mass of PolybHb. ZIF-8P-PolybHb NPs were observed to exhibit an ultrahigh Hb encapsulation efficiency (EE %=93.03.8%) compared to ZIF-8P-Hb NPs (EE %=88.23.5%). The higher Hb encapsulation efficiency could be the result of chelation mediated chemical etching of the ZIF-8P-PolybHb NPs, which generate larger cavities that enable higher protein loading capacity. Additionally, the Hb content of ZIF-8P-PolybHb NPs (4.650.02 mg/mL) was found to be slightly higher than that of ZIF-8P-Hb NPs (4.410.18 mg/mL). ZIF-8P-PolybHb NPs were also observed to have a larger hydrodynamic diameter in comparison to ZIF-8-Hb NPs, which could also result in a larger pore volume.
PolyHb Leakage from ZIF-8P-PolybHb NPs.
[0225] In
Hemocompatibility.
[0226] The interaction between ZIF-8P-PolybHb NPs and RBCs was studied by monitoring the hemolytic activity of the mixture. During hemolysis, Hb tetramers which are composed of two dimers tend to dissociate at relatively low Hb concentration, which can be excreted through the kidney and eventually cause renal failure. Thus, it is important to measure the effect of ZIF-8P-PolybHb NPs on RBC hemolysis. Briefly, ZIF-8P-PolybHb NPs exhibited relatively low hemolysis (3.7%), demonstrating favorable hemocompatibility (<5% hemolysis) according to the ASTM E2524-08 standard.
Encapsulation of PolybHb Reduces the Cytotoxicity of ZIF-8 NPs.
[0227] The in vitro cytotoxicity of ZIF-8P-PolybHb NPs compared to unloaded and bHb loaded ZIF-8 NPs was tested in HUVECs and presented below in
CONCLUSION
[0228] In this Example, we successfully synthesized encapsulated PolybHb into NPs using ZIF-8 precursors (ZIF-8P-PolybHb). Additionally, we established and optimized a continuous-injection method coupled with a chelation-assisted chemical etching process to synthesize ZIF-8P-PolybHb NPs. ZIF-8P-PolybHb NPs were <300 nm in diameter and exhibited O.sub.2 binding properties similar to that of unencapsulated PolybHb. ZIF-8P-PolybHb NPs also exhibited favorable antioxidant (H.sub.2O.sub.2) properties and hemocompatibility, demonstrating its potential as an RBC substitute. ZIF-8P-PolybHb showed no statistically significant impact on HUVEC viability at all concentrations and encapsulation of PolybHb into the ZIF-8 framework improved cell viability compared to unloaded ZIF-8 NPs and bHb loaded ZIF-8 NPs
[0229] All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.