Patent Application
20240366805
This invention is generally in the field of iron oxide nanorods coated with a polymer and methods of making and use thereof.
Iron oxide nanoparticles are widely used magnetic materials for developing molecular imaging probes, in vitro diagnostic agents, image-guided drug delivery, and hyperthermia treatments. In particular, magnetic iron oxide nanorods (IONRs) have gained considerable interest owning to their high relaxivities enhancing magnetic resonance imaging (MRI) contrast. In in vivo applications, anisotropic nanoparticles exhibit favorable pharmacokinetics, higher cell uptake, and improved tumor targeting and intratumoral retention time. In in vitro applications, IONRs with much stronger magnetism allow fast and efficient immunomagnetic separation. However, the use of IONRs in biomedical applications has been hampered by their instability in aqueous medium. The use of coating materials may improve the nanorods' aqueous stability. However, current coated IONRs are limited to those having relatively weak magnetic properties, because IONRs having strong magnetic properties (i.e., a magnetic moment≥10 emu/g, induced using 1 T magnetizing field strength, at room temperature) tend to form clusters in the coating process, which makes coating individual IONR very challenging.
There remains a need for coated iron oxide nanorods that are stable in aqueous medium and have strong magnetic properties and methods for making such.
Therefore, it is the object of the present invention to provide coated iron oxide nanorods that are stable in aqueous medium and have strong magnetic properties.
It is another object of the present invention to provide methods for making the coated iron oxide nanorods.
It is another object of the present invention to provide methods for using the coated iron oxide nanorods.
Described are polymer coated iron oxide nanorods (IONRs), pharmaceutical compositions containing these coated IONRs, and methods of making and using these coated IONRs. The coated IONRs disclosed herein have excellent aqueous stability (i.e., stay suspended in an aqueous medium for at least 1 hour, at room temperature), while maintaining the superior magnetic property of the IONRs (i.e., a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature). This combined features of the disclosed coated IONRs lead to a separation efficiency of these coated IONRs of at least 80%, within 1 min of magnet time, and optionally at least 90%, within 2 min of magnetic time.
The disclosed coated IONRs contain an iron oxide core and a coating. The iron oxide core has a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature. Typically, the iron oxide core of the coated IONRs contains Fe3O4 magnetite or a combination of Fe3O4 magnetite and FeO(OH) goethite. Generally, the iron oxide core has a rod shape, with a length in a range from about 20 nm to about 250 nm and a width in a range from about 2 nm to about 50 nm.
The coating of the coated IONRs can be formed using any suitable materials, such as one or more polymers. In some forms, the coating of the coated IONRs is formed from one or more amphiphilic polymers. For example, the coating of the coated IONRs is formed from an amphiphilic polymer having the structure of Formula I:
In some forms, the polymer can have the structure of Formula II:
For example, the coating of the coated IONRs is formed from a polymer having the structure of Formula III:
When the polymer of any of Formulae I-III forms the coating surrounding the iron oxide core, at least one R is a bond through which a siloxane is attached to the surface of the iron oxide core.
In some forms, the coating of the coated IONRs is formed from a PEG lipid, optionally more than one PEG lipid. PEG lipids are PGE derivatives that are attached to a lipid moiety. For example, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula IV, optionally more than one PEG lipid having the structure of Formula IV:
For example, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula V, optionally more than one PEG lipid having the structure of Formula V:
In some forms, L1 is a bond,
each A1 is independently a bond or
A2 is a bond,
or
Z1 and Z2 are independently a bond or
n3 is an integer from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2, Z+ is a cation (e.g., Na+ or NH4+).
In some forms, L1 is
each A1 is independently a bond or
A2 is a bond,
Z1 and Z2 are independently a bond or
n3 is an integer from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2, Z+ is a cation (e.g., Na+ or NH4+).
In some forms, R1 is
L5 is a bond,
Y1 and Y2 are independently a bond or
n6 and n7 are independently an integer from 1 to 6, R5 an alkyl (e.g., linear, branched, or cyclic C1-C6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tertbutyl, etc.), —SH, —OH, —COOH, —NH2, maleimide, NHS, sulfoNHS, azido, biotin, fluorophore, a clickable group (e.g., BCN, TCO, DBCO, tetrazine, etc.), or a targeting moiety.
In some forms, L2 is
or
In some forms, L3 is
or
Z3 and Z4 are independently a bond or
n8 is an integer from 1 to 6.
In some forms, L4 is hydrogen.
In some forms, L4 is a bond,
or
Z5 and Z6 are independently a bond or
n9 is an integer from 1 to 6.
In some forms, R2 and R3 are independently a C7-C30 or C7-C20 linear alkyl, C7-C30 or C7-C20 linear alkenyl, or C7-C30 or C7-C20 linear alkynyl, optionally substituted with one or more substituent, such as one or more —OH, —COOH, —COO-alkyl, —SH, —NH2, etc.
In some forms, E1 is
n4 and n5 are independently an integer from 1 to 500, from 1 to 200, from 1 to 100, from 1 to 50, from 5 to 1000, from 5 to 500, from 5 to 200, from 5 to 100, from 5 to 50, from 10 to 1000, from 10 to 500, from 10 to 200, from 10 to 100, from 10 to 50, from 20 to 1000, from 20 to 500, from 20 to 200, or from 20 to 100, such as from 20 to 50.
In some forms, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula IV as described above and a PEG lipid having the structure of Formula V as described above.
For example, the coating of the coated IONRs is formed from any one of the PEG lipids shown below, or a mixture of two or more PEG lipids shown below:
where n is an integer from 20 to 50. The coated IONRs have hydrodynamic dimensions, which can be measured using methods known in the art, such as dynamic light scattering. Generally, the coated IONRs have a hydrodynamic length in a range from about 50 nm to about 300 nm, from about 100 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 300 nm, from about 200 nm to about 300 nm, or from about 220 nm to about 300 nm, and a hydrodynamic width in a range from about 20 nm to about 150 nm, from about 20 nm to about 120 nm, from about 20 to about 100 nm, or from about 50 nm to about 150 nm. Further, in some forms, the coated IONRs have a zeta potential in a range from about −10 mV to about −100 mV measured in double distilled water.
Kits and devices containing the coated IONR(s) are disclosed. The kit includes a device and the coated IONR(s). The device in the kit contains one or more defined region(s) or well(s), which are configured to contain a sample for in vitro applications, such as cell separation or target cell detection. Typically, in the kit, the coated IONR(s) are stored separately from the device prior to use. In some forms, the user is provided with just a device. The device contains one or more defined region(s) or well(s) and the coated IONR(s), wherein each defined region or well contains the IONRs.
Methods of producing the disclosed coated iron oxide nanorods (IONRs) are also described. Generally, the method includes (i) mechanically mixing (such as by inverting, shaking, or stirring) a mixture comprising IONRs, a coating material, and a solvent for a time period in a range from about 12 hours to about 60 hours, from about 12 hours to about 48 hours, or from about 24 hours to about 48 hours, at a temperature in a range from 20° C. to about 40° C., such as about 25° C., to form a product comprising coated IONRs; and (ii) during step (i), periodically sonicating the mixture for at least 1 min, at least 2 mins, at least 5 mins, at least 10 mins, at least 20 mins, at least 30 mins, or in a range from 1 min to 1 hour, from 1 min to 30 mins, from 1 min to 20 mins, from 1 min to 10 mins, or from 1 min to 5 mins, at room temperature. Step (ii) can be performed at a regular time interval or irregular time interval during step (i) mechanical stirring the mixture. For example, step (ii) is performed regularly every 30 mins, every hour, or every 2 hours during step (i), or step (ii) is performed irregularly with a time interval of 30 mins, 1 hour, and/or 2 hours.
Optionally, the method further includes one or more of the following steps: dispersing IONRs in the solvent to form a dispersion and sonicating the dispersion for a period in a range from about 1 min to about 1 hour, from about 1 min to about 50 mins, from about 1 min to about 40 mins, from about 1 min to about 30 mins, from about 1 min to about 20 mins, or from about 1 min to about 10 mins, prior to step (i); dissolving the coating material in the solvent to form a solution and adding the solution into the dispersion to form the mixture, prior to step (i); dialyzing the product against deionized water for a period in a range from 1 hour to 60 hours, from 12 hour to 60 hours, from 24 hour to 60 hours, from 12 hour to 48 hours, from 24 hour to 48 hours, from 12 hour to 36 hours, from 12 hour to 24 hours, from 24 hour to 36 hours, subsequent to step (i) and (ii); and collecting the coated IONRs using a magnet and filtering the collected coated IONRs through a filter, optionally wherein the filter has a size of 0.4 μm or less.
The disclosed IONRs can be used in applications such as in purifying target cells, detecting target cells, in vivo or in vitro, delivering active agents, and imaging (as contrast agents).
Polymer coated iron oxide nanorods (also referred to herein as “coated IONRs”) and compositions containing these coated IONRs for in vivo and in vitro applications are described herein. The coated IONRs described herein generally demonstrate stability in an aqueous medium (i.e., stay suspended in the aqueous medium for at least 1 hour, at room temperature) and a strong magnetic property (i.e., a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature). The aqueous stability and superior magnetic property of the disclosed coated IONRs make them suitable for use in a wide range of in vivo and in vitro biomedical applications, such as for use in purifying and detecting one or more target biological substances (such as proteins, peptides, DNAs, RNAs, cells, extracellular vesicles, cytokines, etc.), in vivo or in vitro, delivering active agents, and imaging as contrast agents.
By having the combined properties of aqueous stability and superior magnetic property, the IONRs disclosed herein outperform commercially available iron oxide materials, such as Dynabeads®, the magnetic beads available from StemCell Technology, and Miltenyi Biotec, which only present one of these two properties of the coated IONRs. Coating iron oxide particles with strong magnetic properties is a major limitation in the applications of such materials, because they tend to aggregate and form clusters in the coating process, which results in insufficient coating of these materials and thus poor aqueous stability (for example, Dynabeads® and magnetic beads available from StemCell Technology). To achieve sufficient coating of iron oxide particles, currently available materials use iron oxide particles with relatively weak magnetic property, leading to coated materials with low separation efficiency (for example, magnetic beads available from/supplied by Miltenyi Biotec). These issues are addressed by the coated IONRs disclosed herein. For example, as shown in
When the coated IONRs are tested for their separation performance in vitro, such as by pulling out the coated IONRs dispersed in an aqueous medium using a magnet, the separation efficiency can reach an appreciable level, such as at least 80%, at least 85%, at least 90%, or at least 95%, within 1 min of magnet time. The term “magnet time” refers to the time period of which the coated IONRs are exposed to a magnetic field. The term “separation efficiency” refers to the amount of the dispersed coated IONRs that is pulled out from an aqueous medium by a magnet force using a magnet, relative to the amount of the coated IONRs dispersed in the aqueous medium prior to applying the magnet force, as determined using UV-vis absorbance at room temperature.
The polymer coated IONRs disclosed herein contain an iron oxide core and a coating. The coating surrounds the iron oxide core and may be chemically attached or physically absorbed to the surface of the iron oxide core, or a combination thereof. The disclosed IONRs not only have excellent aqueous stability (i.e., stay suspended in the aqueous medium for at least 1 hour, at room temperature), but also show superior magnetic property (i.e., a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature) and consequently, can achieve a separation efficiency of at least 80% within 1 min of magnet time.
The iron oxide core of the coated IONR has the shape of a rod (also referred to herein as “iron oxide nanorod” or “uncoated iron oxide nanorod”) and provides strong magnetic property (i.e., a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature). For example, the iron oxide core of the coated IONRs has a magnetic moment of at least 20 emu/g, at least 50 emu/g, or at least 80 emu/g, as determined using hysteresis loops, induced using 1 T magnetizing field strength, at room temperature. It is understood that other magnetic materials may be used to replace the iron oxide in the core and then coated with a coating material to form coated nanorods. Examples of other magnetic materials (in place of iron oxide) suitable for forming the core include, but are not limited to, cobalt oxide, nickel oxide, titanium oxide, and ferrites (e.g., ZnFe2O4, SrFe12O19, BaFe12O19, and CoFe2O4).
The iron oxide core can have any suitable dimensions, depending on the specific applications. Generally, the iron oxide core has a length from about 20 nm to about 250 nm and optionally a width from about 2 nm to about 50 nm. For example, the iron oxide core has a length from about 20 nm to <200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, or from about 180 nm to about 250 nm. In some forms, the dimensions of the iron oxide core were measured using a plurality of the core and the dimensions thus refer to the average dimensions. For example, the iron oxide core has an average length from about 20 nm to <200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 250 nm, from about 150 nm to about 200 nm, or from about 180 nm to about 250 nm, and optionally an average width from about 2 nm to about 50 nm. The dimensions of a coated IONR can be measured using methods known in the art, such as transmission electron microscopy (“TEM”) and dynamic light scattering (“DLS”). For example, for measuring the physical dimensions of a coated IONR, TEM is typically used, while for measuring the hydrodynamic dimensions of a coated IONR, DLS is typically used.
The iron oxide in the iron oxide core can be in a variety of forms, such as magnetite (Fe3O4), maghemite (Fe2O3), or goethite (FeO(OH), or a combination thereof. In some forms, the iron oxide in the core is magnetite (Fe3O4). In some other forms, the iron oxide in the core is a combination of magnetite (Fe3O4) and goethite (FeO(OH)). When the iron oxide in the core has more than one form, the content of each form can be in any suitable amount. For example, when the iron oxide in the core is magnetite (Fe3O4), the content of the magnetite can be from about 95% to 100%. For example, when the iron oxide in the core is magnetite (Fe3O4) and goethite (FeO(OH)), the content of the magnetite can be from about 40% to about 80%, such as about 50%, and the remaining balance is goethite (i.e., in a range from about 60% to about 20%, such as bout 50%), for example, about 53% magnetite and about 47% goethite.
The coating is typically formed by a polymer (such as poly(ethylene glycol) (PEG) or a copolymer thereof).
The coating surrounds the iron oxide core and may be chemically attached or physically absorbed to the surface of the iron oxide core, or a combination thereof. In some forms, the coating material, such as PEG or a copolymer thereof, is modified such that it contains one or two terminal moieties at the terminus of the polymer chain. When forming a coating surrounding the iron oxide core, the terminal moiety(ies) of the polymer can chemically attach and/or physically absorbed to the surface of the iron oxide core. Additionally or alternatively, the terminal hydroxyl group(s) of the PEG chain in a coating material can be converted into other functional groups, such as amino, carboxyl, thiol, maleimide, methyl, NHS, sulfoNHS, azide, biotin, clickable, and/or fluorophore groups.
a. Polymers
Examples of polymers that are suitable for forming the coating of the coated IONRs include, but are not limited to, poly(alkylene glycol), such as poly(ethylene glycol), copolymers thereof, and derivatives thereof.
In some forms, the polymer forming the coating of the coated IONRs is a copolymer that contains two or more polymer blocks. When a copolymer is used to form the coating of the coated IONRs, the weight percentage of each polymer block in the copolymer can vary from 5% to 95%. For example, a copolymer containing two different polymer blocks is used to form the coating of the coated IONRs, where the weight percentage of a first polymer block or a second polymer block is in a range from 5% to 95%, such as from 5% to 90%, from 5% to 80%, from 5% to 75%, from 10% to 90%, from 10% to 75%, from 20% to 90%, from 20% to 80%, from 25% to 75%, from 25% to 50%, from 30% to 90%, from 40% to 90%, from 50% to 90%, or from 50% to 75%. The weight percentage of a polymer block refers to the total weight of the blocks containing the same repeating unit in the polymer chain.
In some forms, the coating of the coated IONRs is formed using a blend of two or more polymers. When a blend of polymers is used to form the coating of the coated IONRs, the weight percentage of each polymer in the blend can vary from 5% to 95%. For example, a blend of two polymers is used to form the coating of the coated IONRs, where the weight percentage of a first polymer or a second polymer is in a range from 5% to 95%, such as from 5% to 90%, from 5% to 80%, from 5% to 75%, from 10% to 90%, from 10% to 75%, from 20% to 90%, from 20% to 80%, from 25% to 75%, from 25% to 50%, from 30% to 90%, from 40% to 90%, from 50% to 90%, or from 50% to 75%.
The polymer or each polymer in a blend of polymers that are suitable for forming the coating of the coated IONRs can have a molecular weight in a range from 1.5 kDa to 300 kDa, from 1.5 kDa to 275 kDa, from 1.5 kDa to 250 kDa, from 1.5 kDa to 100 kDa, from 2 kDa to 80 kDa, from 2 kDa to 50 kDa, from 2 kDa to 30 kDa, from 2 kDa to 20 kDa, or from 2 kDa to 10 kDa.
Generally, the polymer or blend of polymers, regardless of its/their composition (i.e. homo- or poly-), used for forming the coating of the coated IONRs is hydrophilic or amphiphilic. Whether a polymer or blend of polymers is hydrophilic can also be determined via contact angle. For example, if a polymer or blend of polymers is applied to a surface, such as glass, and forms a contact angle with water, which is smaller than the contact angle of water on a surface of glass without the polymer or blend of polymers, the polymer or blend of polymers is hydrophilic.
In preferred forms, the polymer forming the coating of the coated IONRs is an amphiphilic polymer containing one or more PEG block(s) and one or more hydrophobic polymer block(s), such as the diblock copolymer PEG-b-AGE as described in U.S. Pat. No. 10,393,736 (described in detail below) and PEG lipids. In some forms, when forming a coating on the iron oxide core, the amphiphilic polymer is arranged such that the hydrophilic polymer block(s) form(s) the outer layer of the coating and the hydrophobic polymer block(s) form(s) the inner layer of the coating. In some forms, the hydrophobic polymer block is at one of the terminus of the amphiphilic polymer chain and the hydrophobic terminus is modified to contain a terminal moiety, through which the amphiphilic polymer is attached to the surface of the iron oxide core. In some forms, the amphiphilic polymer contains one or more targeting moieties, wherein the targeting moieties are arranged such that they are exposed to a surrounding environment when the amphiphilic polymer forms the coating on the iron oxide core.
i. Exemplary Polymers
A preferred polymer for forming the coating of the coated IONRs is a diblock copolymer (PEG-b-AGE) that contains aminated hydrophilic PEG chains and hydrophobic allyl glycidyl ether (AGE) moieties. However, the terminal functional groups of the PEG-b-AGE are not limited to amino groups. For example, one or all of the amino groups of PEG of the PEG-b-AGE can be alternatively carboxyl, thiol, maleimide, methyl, NHS, sulfoNHS, azido, biotin, clickable, or fluorophore groups, or a combination thereof. The PEG-b-AGE can provide excellent anti-biofouling property when coated on iron oxide spherical nanoparticles; however, due to the strong magnetic property of the iron oxide nanorods disclosed herein, the coating method disclosed in U.S. Pat. No. 10,393,736 does not result in PEG-b-AGE coated IONRs having the aqueous stability and superior magnetic property disclosed herein.
In some forms, the polymer can have the structure of Formula I:
In some forms, the polymer can have the structure of Formula II:
In some forms, R″ of Formulae I and II can be hydrogen, a substituted or unsubstituted alkyl, a substituted or unsubstituted phenyl, or a substituted or unsubstituted heteroaryl. In some forms, R″ of Formulae I and II can be hydrogen, an unsubstituted alkyl, an unsubstituted phenyl, or an unsubstituted heteroaryl. In some forms, R″ of Formulae I and II can be hydrogen.
When one or more functional groups of the polymer is/are alkyl(s), the alkyl can be independently a linear alkyl, a branched alkyl, or a cyclic alkyl (either monocyclic or polycyclic), substituted or unsubstituted. The terms “cyclic alkyl” and “cycloalkyl” are used interchangeably herein. Exemplary alkyl include a linear C1-C30 alkyl, a branched C4-C30 alkyl, a cyclic C3-C30 alkyl, a linear C1-C20 alkyl, a branched C4-C20 alkyl, a cyclic C3-C20 alkyl, a linear C1-C10 alkyl, a branched C4-C10 alkyl, a cyclic C3-C10 alkyl, a linear C1-C6 alkyl, a branched C4-C6 alkyl, a cyclic C3-C6 alkyl, a linear C1-C4 alkyl, cyclic C3-C4 alkyl, such as a linear C1-C10, C1-C9, C1-C8, C1-C7, C1-C6, C1-C5, C1-C4, C1-C3, or C1-C2 alkyl group, a branched C3-C9, C3-C9, C3-C8, C3-C7, C3-C6, C3-C5, or C3-C4 alkyl group, or a cyclic C3-C9, C3-C9, C3-C8, C3-C7, C3-C6, C3-C5, or C3-C4 alkyl group. The cyclic alkyl can be a monocyclic or polycyclic alkyl, such as a C4-C30, C4-C25, C4-C20, C4-C18, C4-C16, C4-C15, C4-C14, C4-C13, C4-C12, C4-C10, C4-C9, C4-C8, C4-C7, C4-C6, or C4-C5 monocyclic or polycyclic alkyl group. When the alkyl is a substituted alkyl, each substituent can be an unsubstituted alkyl, an unsubstituted alkenyl, an unsubstituted alkynyl, an unsubstituted heterocyclyl, an unsubstituted aryl, an unsubstituted heteroaryl, an unsubstituted polyaryl, an unsubstituted polyheteroaryl, an unsubstituted aralkyl (e.g. benzyl), a carbonyl, an alkoxy (e.g. methoxy, ethoxy, aryloxy, benzoether, etc.), a halide, a hydroxyl, or a haloalkyl (e.g. —CH2Br, —CF3, etc.).
When one or more functional groups of the polymer is/are aryl(s), the aryl group can be independently a C5-C30 aryl, a C5-C20 aryl, a C5-C12 aryl, a C5-C11 aryl, a C5-C9 aryl, a C6-C20 aryl, a C6-C12 aryl, a C6-C11 aryl, or a C6-C9 aryl. It is understood that the aryl can be a heteroaryl, such as a C5-C30 heteroaryl, a C5-C20 heteroaryl, a C5-C12 heteroaryl, a C5-C11 heteroaryl, a C5-C9 heteroaryl, a C6-C30 heteroaryl, a C6-C20 heteroaryl, a C6-C12 heteroaryl, a C6-C11 heteroaryl, or a C6-C9 heteroaryl. When the aryl is a substituted aryl, each substituent can be an unsubstituted alkyl, an unsubstituted alkenyl, an unsubstituted alkynyl, an unsubstituted heterocyclyl, an unsubstituted aryl, an unsubstituted heteroaryl, an unsubstituted polyaryl, an unsubstituted polyheteroaryl, an unsubstituted aralkyl (e.g. benzyl), a carbonyl, an alkoxy (e.g. methoxy, ethoxy, aryloxy, benzoether, etc.), a halide, a hydroxyl, or a haloalkyl (e.g. —CH2Br, —CF3, etc.).
When one or more functional groups of the polymer is/are heteroaryl(s), the heteroaryl can be independently a C5-C26-membered aromatic or fused aromatic ring system, in which one or more carbon atoms on one or more aromatic ring structures have been substituted with a heteroatom. Suitable heteroatoms include, but are not limited to, oxygen, sulfur, and nitrogen. Examples of heteroaryl groups pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, tetrazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Examples of heteroaryl rings include, but are not limited to, benzimidazolyl, benzofuranyl, benzothiofuranyl, benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl, benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl, benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl, chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl, furanyl, furazanyl, imidazolidinyl, imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl, indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl, isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl, isothiazolyl, isoxazolyl, methylenedioxyphenyl, naphthyridinyl, octahydroisoquinolinyl, 1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, oxazolidinyl, oxazolyl, oxindolyl, pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl, pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl, pyrimidinyl, pyrrolidinyl, pyrrolinyl, pyrrolyl, quinazolinyl, quinolinyl, quinoxalinyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl, tetrazolyl, 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl, thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl, thienoimidazolyl, thiophenyl and xanthenyl. One or more of the rings can be substituted as defined above for “substituted heteroaryl.”
When the polymer of Formula I or II forms a coating surrounding the iron oxide core, one or more of the R groups is a bond through which a siloxane is attached to the surface of the iron oxide core. A “siloxane” refers to a moiety of silicon covalently bound to oxygen.
In some forms, the polymer can have the structure of Formula III:
The linking moiety L′ of Formula I and L1 and L2 of Formulae II-III can be any suitable molecular arrangements for bridging two molecular moieties together. An example formula is —(R″)b—, wherein b can be an integer from 1 to 10, R″ can be individually and independently, at each occurrence, —CR1R2-, —CHR1-, —CH2—, —C(OH)R1-, —C(OH)(OH)—, —C(OH)H—, —C(CN)R1-, —C(CN)(CN)—, —C(CN)H—, —NR1-, —NH—, —S—, —S—S—, —O—, —(C═CH2)—, —(C═NH)—, —(C═S)—, —(C═O)—, —(C═O)O—, —O(C═O)—, and —(C═O)CH(CH3)—, and R1 and R2 can be independently a hydrogen, a halogen, an alkenyl, an hydroxyl, a thiol, an amino, an azide, a cyano, or an alkyl as defined above for R and R′.
In some forms, at least one T′ of Formula I is a targeting moiety. The targeting moiety can be any suitable molecules (such as NTA-Ni) or entity capable of binding a biological substance, such as proteins (e.g., antibodies), peptide, extracellular vesicles, or a surface marker that is expressed by a specific cell population, e.g., lymphocytes, monocytes, stem cells, or tumor cells. Examples of targeting moieties include, but are not limited to, an antibody, an antibody fragment, an antibody mimetic, an affibody, a nucleic acid, an oligonucleotide, an aptamer, a peptide, a steroid, and a tyrosine kinase inhibitor. The targeting moiety may be directly conjugated to A′ in Formula I, or conjugated to the nitrogen via a linking moiety L′ as defined above. More specific examples of suitable targeting moieties are described in U.S. Pat. No. 10,393,736, the disclosure of which is incorporated herein by reference in its entirety. In forms where each occurrence of T′ is hydrogen, a targeting moiety such as any one of those described herein can be conjugated to the terminal functional groups, such as amino, carboxyl, thiol, and/or maleimide, of the polymer after the polymer is coated on the IONR core.
In some forms, each occurrence of L′ can be independently a linking moiety as defined above and T′ can be hydrogen. In some forms, each occurrence of L′ can be independently a single bond and T′ can be hydrogen. In some forms, a first L′ can be a single bond, a second L′ can be a linking moiety as defined above, a first T′ can be hydrogen, and a second T′ can be a targeting moiety as defined above.
In some forms, at least one of T1 and T2 of any of Formulae II-III is a targeting moiety. The targeting moiety can be anyone of those described above for T′. In forms where T1 and T2 of any of Formulae II-III are hydrogen, a targeting moiety such as any one of those described herein can be conjugated to the terminal amino groups of the polymer after the polymer is coated on the IONR core.
In some forms, for any of Formulae II-III, L1 and L2 can be independently a linking moiety as defined above and T1 and T2 can be hydrogen. In some forms, for any of Formulae II-III, L1 and L2 can be independently a single bond and T1 and T2 can be hydrogen. In some forms, for any of Formulae II-III, L1 can be a single bond, L2 can be a linking moiety as defined above, T1 can be hydrogen, and T2 can be a targeting moiety as defined above.
Another preferred polymer for forming the coating of the coated IONRs is PEG lipid. For example, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula IV, optionally more than one PEG lipid having the structure of Formula IV:
n6 and n7 are independently an integer from 1 to 6, X4-X6 are independently O or S, R5 is hydrogen, an alkyl (e.g., linear, branched, or cyclic C1-C6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tertbutyl, etc.), —SH, —OH, —COOH, COR6, —COOR6, —NH2, maleimide, NHS, sulfoNHS, azido, biotin, fluorophore, aldehyde, biomolecule (e.g., peptide, protein, protein fragment, etc.), vinylsulfone, a clickable group (e.g., BCN, TCO, DBCO, tetrazine, etc.), or a targeting moiety as described above, R6 is an alkyl, —NH2, maleimide, NHS, sulfoNHS, azido, biotin, fluorophore, aldehyde, biomolecule (e.g., peptide, protein, protein fragment, etc.), vinylsulfone, a clickable group (e.g., BCN, TCO, DBCO, tetrazine, etc.), or a targeting moiety as described above; (v) L2 is a bond,
For example, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula V, optionally more than one PEG lipid having the structure of Formula V:
In some forms, L1 is a bond,
each A1 is independently a bond or
A2 is a bond,
or
Z1 and Z2 are independently a bond or
n3 is an integer from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2, Z+ is a cation (e.g., Na+ or NH4+).
In some forms, L1 is
each A1 is independently a bond or
A2 is a bond,
Z1 and Z2 are independently a bond or
n3 is an integer from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, or from 1 to 2, Z+ is a cation (e.g., Na+ or NH4+).
In some forms, R1 is
L5 is a bond,
Y1 and Y2 are independently a bond or
n6 and n7 are independently an integer from 1 to 6, R5 an alkyl (e.g., linear, branched, or cyclic C1-C6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, tertbutyl, etc.), —SH, —OH, —COOH, —NH2, maleimide, NHS, sulfoNHS, azido, biotin, fluorophore, a clickable group (e.g., BCN, TCO, DBCO, tetrazine, etc.), or a targeting moiety.
In some forms, L2 is
In some forms, L3 is
or
Z3 and Z4 are independently a bond or
n8 is an integer from 1 to 6.
In some forms, L4 is hydrogen.
In some forms, L4 is a bond,
Z5 and Z6 are independently a bond or
n9 is an integer from 1 to 6.
In some forms, R2 and R3 are independently a C7-C30 or C7-C20 linear alkyl, C7-C30 or C7-C20 linear alkenyl, or C7-C30 or C7-C20 linear alkynyl, optionally substituted with one or more substituent, such as one or more —OH, —COOH, —COO-alkyl, —SH, —NH2, etc.
In some forms, E1 is
n4 and n5 are independently an integer from 1 to 500, from 1 to 200, from 1 to 100, from 1 to 50, from 5 to 1000, from 5 to 500, from 5 to 200, from 5 to 100, from 5 to 50, from 10 to 1000, from 10 to 500, from 10 to 200, from 10 to 100, from 10 to 50, from 20 to 1000, from 20 to 500, from 20 to 200, or from 20 to 100, such as from 20 to 50.
In some forms, the coating of the coated IONRs is formed from a PEG lipid having the structure of Formula IV as described above and a PEG lipid having the structure of Formula V as described above.
For example, the coating of the coated IONRs is formed from any one of the PEG lipids shown below, or a mixture of two or more PEG lipids shown below:
where n is an integer from 20 to 50.
b. Active Agents
Optionally, the coated IONRs contain one or more active agents embedded in the coating. The active agent(s) embedded in the coating of the coated IONRs may be hydrophilic or hydrophobic, depending on the components and the structure of the coating. For example, in some forms, the coating is formed by one or more amphiphilic polymer(s), wherein the outer layer of the coating is formed by the hydrophilic polymer block(s) of the amphiphilic polymer and the inner layer of the coating is formed by the hydrophobic polymer block(s) of the amphiphilic polymer. In these forms, the active agents contained in the coated IONRs are typically hydrophobic and embedded in the hydrophobic inner layer of the coating.
The active agent or each active agent of two or more active agents within the coated IONRs can be a therapeutic agent, a diagnostic agent, or a prophylactic agent. Active agents with a wide range of molecular weight can be loaded in the coated IONRs, for example, between 100 Da and 10,000 kDa. Examples of active agents that can be loaded in the coated IONRs for delivery include, but are not limited to, small molecules, proteins, polypeptides, peptides, carbohydrates, nucleic acids, glycoproteins, lipids, and antibodies/antigens, and combinations thereof. “Small molecule” generally refers to an organic molecule that is less than about 900 Da. Typically, small molecules are non-polymeric and/or non-oligomeric.
The weight percentage of the active agent or the total weight percentage of the two or more active agents in the coated IONRs, when present, depends on the hydrophilicity/hydrophobicity and the molecular weights of the active agents. Optionally, the weight percentage of the active agent or the total weight percentage of the two or more active agents in the coated IONRs is in a range from 0.01% to 80%, from 0.01% to 60%, from 0.01% to 50%, from 0.01% to 40%, from 0.01% to 25%, from 0.01% to 10%, from 0.01% to 5%, from 0.1% to 50%, from 0.1% to 25%, from 0.1% to 10%, from 0.1% to 5%, from 0.05% to 10%, from 0.05% to 5%, from 0.01% to 2%, from 0.05% to 2%, from 0.1% to 2%, from 0.01% to 1%, from 0.05% to 1%, or from 0.1% to 1%. The term “weight percentage of the active agent” refers to the weight of the active agent relative to the sum of the weights of the active agent, the coating, and the core. The term “total weight percentage of the two or more active agents” refers to the sum of the weights of the active agents relative to the sum of the weights of the active agents, the coating, and the core.
The weight percentage of the active agent or total weight percentage of the two or more active agents can be varied based on the specific agent(s) being delivered. For example, for large biomolecules, such as proteins and nucleic acids, typical weight percentages of the large biomolecule in the coated IONRs are from 0 0.01% to 20%, from 0.01% to 5%, from 0.01% to 2.5%, or from 0.01% to 1%.
Examples of active agents and their alternative forms such as alternative salt forms, free acid forms, free base forms, and hydrates that can be loaded in the coating of the coated IONRs for delivery include, but are not limited to, anticancer agents (such as those described in U.S. Pat. No. 10,393,736, the disclosure of which is incorporated herein by reference in its entirety); analgesics/antipyretics (such as aspirin, acetaminophen, ibuprofen, etc.); antibiotics; antidepressants; antidiabetics; antifungal agents; antihypertensive agents; anti-inflammatories; antianxiety agents; immunosuppressive agents; antimigraine agents; sedatives/hypnotics; antianginal agents; antipsychotic agents; antimanic agents; antiarrhythmics; antiarthritic agents; antigout agents; anticoagulants; thrombolytic agents; antifibrinolytic agents; hemorheologic agents; antiplatelet agents; anticonvulsants; antiparkinson agents; antihistamines/antipruritics; agents useful for calcium regulation; antibacterial agents; antiviral agents; antimicrobials; anti-infectives; bronchodilators; steroidal compounds, hormones and hormone analogues; hypoglycemic agents; hypolipidemic agents; peptides, proteins, and nucleic acids; agents useful for erythropoiesis; antiulcer/anti-reflux agents; antinauseants/antiemetics; and vitamins. Any of these active agents or a combination thereof can be loaded in the coated IONRs for delivery.
In some forms, the active agent(s) loaded in the coating of the coated IONRs for delivery is/are anticancer agent(s), such as temozolomide, carmustine, bevacizumab, procarbazine, lomustine, vincristine, gefitinib, erlotinib, cisplatin, carboplatin, oxaliplatin, 5-fluorouracil, gemcitabine, tegafur, raltitrexed, methotrexate, cytosine arabinoside, hydroxyurea, adriamycin, bleomycin, doxorubicin, daunomycin, epirubicin, idarubicin, mitomycin-C, dactinomycin, mithramycin, vinblastine, vindesine, vinorelbine, paclitaxel, taxol, docetaxel, etoposide, teniposide, amsacrine, topotecan, camptothecin, bortezomib, anagrelide, tamoxifen, toremifene, raloxifene, droloxifene, iodoxyfene, fulvestrant, bicalutamide, flutamide, nilutamide, cyproterone, goserelin, leuprorelin, buserelin, megestrol, anastrozole, letrozole, vorozole, exemestane, finasteride, marimastat, trastuzumab, cetuximab, dasatinib, imatinib, combretastatin, thalidomide, azacitidine, azathioprine, capecitabine, chlorambucil, cyclophosphamide, cytarabine, daunorubicin, doxifluridine, epothilone, irinotecan, mechlorethamine, mercaptopurine, mitoxantrone, pemetrexed, tioguanine, valrubicin and/or lenalidomide or combinations thereof such as cyclophosphamide, methotrexate, 5-fluorouracil (CMF); doxorubicin, cyclophosphamide (AC); mustine, vincristine, procarbazine, prednisolone (MOPP); sdriamycin, bleomycin, vinblastine, dacarbazine (ABVD); cyclophosphamide, doxorubicin, vincristine, prednisolone (CHOP); bleomycin, etoposide, cisplatin (BEP); epirubicin, cisplatin, 5-fluorouracil (ECF); epirubicin, cisplatin, capecitabine (ECX); and methotrexate, vincristine, doxorubicin, cisplatin (MVAC); and combinations thereof.
The performance of the coated IONRs can be evaluated by the properties of these materials, such as hydrodynamic dimensions, zeta potential, magnetic properties, dispersion in aqueous medium, and separation efficiency.
a. Hydrodynamic Dimensions and Zeta Potential
The hydrodynamic dimensions of the coated IONRs reflects the hypothetical dimensions of an IONR moving in the solution. The coated IONRs typically have a hydrodynamic length and a hydrodynamic width, and can be measured in an aqueous medium using dynamic light scattering. Generally, the coated IONRs have a hydrodynamic length in a range from about 50 nm to about 300 nm, from about 100 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 300 nm, from about 200 nm to about 300 nm, or from about 220 nm to about 300 nm, and a hydrodynamic width in a range from about 20 nm to about 150 nm, from about 20 nm to about 120 nm, from about 20 to about 100 nm, or from about 50 nm to about 150 nm.
In some forms, the hydrodynamic dimensions of the coated IONR are measured using a plurality of coated IONRs and thus reflect the average of the hydrodynamic dimensions of the plurality of coated IONRs. In these forms, the coated INOR can have an average hydrodynamic length in a range from about 50 nm to about 300 nm, from about 100 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 300 nm, from about 200 nm to about 300 nm, or from about 220 nm to about 300 nm, and an average hydrodynamic width in a range from about 20 nm to about 150 nm, from about 20 nm to about 120 nm, from about 20 to about 100 nm, or from about 50 nm to about 150 nm.
The physical dimensions, i.e., the length and width, of the coated IONRs can be measured using the TEM, which measures the actual dimensions of the coated IONRs in solid state. Generally, the coated IONRs have an aspect ratio (length/width) ranging from 2 to 20, from 5 to 20, from 2 to 15, from 2 to 10, from 5 to 15, from 5 to 10, or from 6 to 9.
The material forming the coating of the coated IONRs can be positively charged, negatively charged, or have a near-neutral charge, depending on the types and numbers of terminal functional groups. In some forms, the material forming the coating of the coated IONRs is negatively charged or contain(s) one or more moieties (i.e. one or more functional groups, for example, amino groups) that impart a negative charge to the coating material in water. In these forms, when the iron oxide core is coated, the coating surrounding the core can provide a negative zeta potential for the coated IONR, measured in double distilled water at room temperature using a Zetasizer or similar instrument such as Zetaview. For example, the coated IONRs can have a zeta potential in a range from about −10 mV to −100 mV, in DI water at room temperature. The term “room temperature” refers to a temperature in a range from 20° C. to 25° C., such as about 25° C., at 1 atm.
An uncoated IONRs (i.e., iron oxide core) cannot be dispersed in aqueous medium sufficiently long for measurements of hydrodynamic dimensions and zeta potential. Thus, a successful measurement of hydrodynamic dimensions and/or zeta potential of the material in an aqueous medium, such as double distilled water, is a way of demonstrating that a coating formed around the iron oxide core.
b. Magnetic Properties
The magnetic properties of the coated IONRs can be determined using hysteresis curve, measured by a superconducting quantum interference device (SQUID). A hysteresis curve shows the magnetic moment (, emu/g) of the material being measured at different applied magnetizing field strength. A magnetic moment≥10 emu/g, induced using 1 T magnetizing field strength, at room temperature, is considered as having strong magnetic property. The coated IONRs typically have a magnetic moment of at least 10 emu/g, at least 20 emu/g, at least 50 emu/g, at least 80 emu/g, induced using 1 T magnetizing field strength, at room temperature. Such a magnetic moment of the disclosed coated IONRs is significantly stronger than those disclosed in previous studies, see, e.g., U.S. Pat. No. 9,125,941. For example, the PEG-b-AGE coated IONRs have magnetic moment of at least 50 emu/g or at least 75 emu/g, induced using 1 T magnetizing field strength, at room temperature.
c. Aqueous Stability
Self-precipitation of IONRs in aqueous medium is a main issue for their use in biomedical application. The coated IONRs disclosed herein have excellent aqueous stability. For example, the coated IONRs can stay suspended in the aqueous medium for at least 1 hour, at least 2 hours, at least 4 hours, at least 12 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 5 days, or at least 7 days, at room temperature, as determined using a UV-vis spectrometer. Compared to uncoated IONRs, the coated IONRs can stay suspended in the aqueous medium for a time period that is at least 50-time longer, at least 100-time longer, at least 200-time longer, or at least 1000-time longer, at room temperature, under the same measurement conditions.
In some forms, the aqueous stability of the coated IONRs is measured by dispersing a plurality of the coated IONRs in an aqueous medium and measuring the UV-vis change at different time point to determine the amount percentage of coated IONRs that remain suspended in the aqueous medium, relative to the amount of coated IONRs at time zero. Time zero is the UV-vis measurement made immediately (within 1 min) after dispersing the coated IONRs in the aqueous medium. For example, at least 90%, at least 95%, or at least 98% of the coated IONRs remain suspended in the aqueous medium at 4 hours, at room temperature, as determined using a UV-vis spectrometer. For example, at least 75%, at least 80%, or at least 85% of the coated IONRs that remain suspended in the aqueous medium at 48 hours, for 3 days, or for 7 days, at room temperature, as determined using a UV-vis spectrometer. Compared to uncoated IONRs, the amount/percentage of coated IONRs that remain suspended in the aqueous medium is at least 90% more, at least 95% more, or 100% more, at 4 hours, at room temperature, under the same measurement conditions; and/or at least 75%, at least 80%, or at least 85% more, at 48 hours, for 3 days, or for 7 days, at room temperature, under the same measurement conditions.
In some forms, the aqueous stability of the coated IONRs disclosed herein is superior to that of the commercially available magnetic beads, such as Dynabead® (Lot #: 00747022), of the magnetic beads of supplied by StemCell Technology (Lot #: 1000096771), and/or Miltenyi Biotec (Lot #: 5221009844). For example, the coated IONRs can stay suspended in the aqueous medium for a period that is at least 50-time longer, 100-time longer, or 200-time longer, compared to Dynabead® (Lot #: 00747022), magnetic beads supplied by StemCell Technology (Lot #: 1000096771), and/or Miltenyi Biotec (Lot #: 5221009844), at room temperature, under the same measurement conditions. For example, the amount/percentage of coated IONRs that remain suspended in the aqueous medium is at least 40%, at least 60%, or at least 90% more than the amount/percentage of Dynabead® (Lot #: 00747022) and/or the magnetic beads supplied by StemCell Technology (Lot #: 1000096771), at room temperature, under the same measurement conditions; and/or at least 5%, at least 10%, at least 15%, or at least 20% more than the amount/percentage of the magnetic beads supplied by Miltenyi Biotec (Lot #: 5221009844), at room temperature, under the same measurement conditions.
An exemplary method of determining whether particles are suspended in an aqueous medium using UV-vis spectrometer is described below. For example, a solution of coated IONRs is vortexed and then set aside undisturbed to allow slow precipitation of coated IONRs out of suspension, if any. At different time points during setting, ranging from 1 min to 7 days, aliquots of the bulk solution are pipetted onto a 96 well plate, which is measured at 400 nm to obtain the absorbance values. The stability of the IONRs is calculated by comparing the absorbance at each time point vs that of the bulk solution taken after vortexing.
d. Separation Efficiency
In addition to the excellent aqueous stability described above, the disclosed coated IONRs also show superior in vitro separation efficiency. The separation efficiency can be measured by pulling out coated IONRs dispersed in an aqueous medium using a magnet, and the value is determined using the percentage of a plurality of dispersed coated IONRs that was pulled out from the aqueous medium within a short period of time, such as within 1 min of magnet time. Typically, the separation efficiency of the coated IONRs is at least 80%, at least 85%, at least 90%, or at least 95%, within 1 min of magnet time.
In some forms, the separation efficiency of the coated IONRs disclosed herein is higher than that of the commercially available magnetic beads, such as the magnetic beads of Miltenyi Biotec (Lot #: 5221009844). For example, the coated IONRs have a separation efficiency that is at least 10-time higher, 15-time longer, or 20-time higher, compared to the magnetic beads of Miltenyi Biotec (Lot #: 5221009844), under the same measurement conditions.
Pharmaceutical compositions that contain a plurality of the coated IONRs described herein in a form suitable for in vitro or in vivo applications (such as administration to a mammal), are disclosed. The pharmaceutical composition may include one or more pharmaceutically acceptable carriers and/or one or more pharmaceutically acceptable excipients. For example, the pharmaceutical composition may be in the form of a liquid, such as a solution or a suspension, and contain a plurality of the disclosed coated IONRs in an aqueous medium and, optionally, one or more suitable excipients for the liquid composition. Optionally, the pharmaceutical composition is in a solid form, and contains a plurality of the disclosed coated IONRs and one or more suitable excipients for a solid composition.
Suitable pharmaceutically acceptable carriers and excipients are generally recognized as safe (GRAS), and may be administered to an individual without causing undesirable biological side effects or unwanted interactions.
Representative carriers and excipients include solvents (including buffers), diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, and stabilizing agents, and a combination thereof.
Excipients can be added to a liquid or solid pharmaceutical composition (for in vivo or in vitro applications) to assist in sterility, stability (e.g. shelf-life), integration, and to adjust and/or maintain pH or isotonicity of the coated IONRs in the pharmaceutical composition, such as diluents, pH modifying agents, preservatives, antioxidants, suspending agents, wetting agents, viscosity modifiers, tonicity agents, and stabilizing agents, and a combination thereof.
Coated IONRs for administering active agents to a subject in need thereof, such as a mammal, can be dissolved or suspended in a suitable carrier to form a liquid pharmaceutical formulation, such as sterile saline, phosphate buffered saline (PBS), balanced salt solution (BSS), viscous gel, or other pharmaceutically acceptable carriers for administration. The pharmaceutical formulation may also be a sterile solution, suspension, or emulsion in a nontoxic, parenterally acceptable diluent or solvent.
A plurality of the disclosed coated IONRs can be formulated into a pharmaceutical formulation, in a liquid form or a solid form, as a liquid formulation or a solid formulation for oral administration or parenteral administration (e.g. intramuscular administration, intravenous administration, intraperitoneal administration, and subcutaneous administration) to a subject.
a. Oral Formulations
The pharmaceutical formulation containing a plurality of the disclosed coated IONRs may be provided in a form suitable for oral administration to a subject, such as a mammal (i.e., an oral composition). Oral administration may involve swallowing, so that coated IONRs, optionally encapsulating active agent(s) in the coating, enter the gastrointestinal tract, or buccal or sublingual administration may be employed by which the coated IONRs encapsulating active agent(s) enter the blood stream directly from the mouth.
Formulations suitable for oral administration include solid formulations such as tablets, capsules containing particulates, liquids, powders, lozenges (including liquid-filled lozenges), chews, multi- and nano-particulates, gels, solid solutions, liposomes, films, ovules, sprays, and liquid formulations.
Liquid formulations for oral administration include suspensions, solutions, syrups, and elixirs. Such oral formulations may be employed as fillers in soft or hard capsules and can contain one or more suitable carriers and/or excipients, for example, water, ethanol, polyethylene glycol, propylene glycol, chitosan polymers and chitosan derivatives (e.g. N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), methylcellulose, a suitable oil, one or more emulsifying agents, and/or suspending agents. Liquid formulations for oral administration may also be prepared by the reconstitution of a solid, for example, from a sachet.
Optionally, the coated IONRs are included in a fast-dissolving and/or fast-disintegrating dosage form.
For tablet or capsule dosage forms, in addition to the coated IONRs described herein, tablets generally contain disintegrants, binders, diluents, surface active agents, lubricants, glidants, antioxidants, colourants, flavoring agents, preservatives, or taste masking agents, or a combination thereof.
Examples of suitable disintegrants for forming a table or capsule dosage form containing the coated IONRs include, but are not limited to, sodium starch glycolate, sodium carboxymethyl cellulose, calcium carboxymethyl cellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone, methyl cellulose, microcrystalline cellulose, lower alkyl-substituted hydroxypropyl cellulose, starch, pregelatinised starch and sodium alginate. Generally, the disintegrant can have a concentration in a range from about 1 wt % to about 25 wt %, from about 5 wt % to about 20 wt % of the tablet or capsule dosage form containing the coated IONRs.
Binders are generally used to impart cohesive qualities to a tablet formulation containing the coated IONRs. Suitable binders for forming a tablet or capsule formulation containing the coated IONRs include, but are not limited to, microcrystalline cellulose, gelatin, sugars, polyethylene glycol, natural and synthetic gums, polyvinylpyrrolidone, pregelatinised starch, chitosan polymers and chitosan derivatives (e.g. N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), hydroxypropyl cellulose, and hydroxypropyl methylcellulose.
Suitable diluents for forming a table or capsule formulation containing the coated IONRs include, but are not limited to, lactose (as, for example, the monohydrate, spray-dried monohydrate or anhydrous form), chitosan polymers and chitosan derivatives (e.g. N-trimethylene chloride chitosan, chitosan esters, chitosan modified with hydrophilic groups, such as amino groups, carboxyl groups, sulfate groups, etc.), N-sulfonated derivatives of chitosan, quaternarized derivatives of chitosan, carbosyalkylated chitosan, microcrystalline chitosan, mannitol, xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starch and dibasic calcium phosphate dihydrate.
Tablet or capsule formulation containing the coated IONRs may also contain surface active agents, such as sodium lauryl sulfate and polysorbate 80, and glidants such as silicon dioxide and talc, in the coating. When present, surface active agents can have a concentration in a range from about 0.2 wt % to 5 wt % of the tablet or capsule formulation.
Tablet or capsule formulations containing the coated IONRs also generally contain lubricants, such as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate, and mixtures of magnesium stearate with sodium lauryl sulphate. Lubricants can have a concentration in a range from about 0.25 wt % to 10 wt %, from about 0.5 wt % to about 3 wt % of the tablet or capsule formulation.
Other possible excipients included in a tablet or capsule formulation containing the coated IONRs include glidants (e.g. Talc or colloidal anhydrous silica at about 0.1 wt % to about 3 wt % of the table or capsule formulation), antioxidants, colourants, flavouring agents, preservatives and taste-masking agents. When present, glidants can have a concentration in a range from about 0.2 wt % to 1 wt % of the tablet or capsule formulation.
An exemplary tablet formulation contains up to about 80 wt % of the coated IONRs described herein, from about 10 wt % to about 90 wt % binder, from about 0 wt % to about 85 wt % diluent, from about 2 wt % to about 10 wt % disintegrant, and from about 0.25 wt % to about 10 wt % lubricant.
Tablet or capsule blends, including the coated IONRs and one or more suitable excipients, may be compressed directly or by roller to form tablets. Tablet or capsule blends or portions of the blends may alternatively be wet-, dry-, or melt-granulated, melt congealed, or extruded before tableting. The final table or capsule formulation may contain one or more layers and may be coated or uncoated; it may even be encapsulated in a particle, such as a polymeric particle or a liposomal particle.
Solid formulations containing the coated IONRs for oral administration may be formulated to be immediate and/or modified release. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.
b. Parenteral Formulations
Optionally, the pharmaceutical formulation containing a plurality of the disclosed coated IONRs is in a form suitable for administration directly into the blood stream, into muscle, or into an internal organ. Suitable routes for such parenteral administration include intravenous, intraarterial, intraperitoneal, intrathecal, epidural, intracerebroventricular, intraurethral, intrasternal, intracranial, intramuscular, and subcutaneous delivery. Suitable means for parenteral administration include needle (including microneedle) injectors, needle-free injectors, and infusion techniques.
For example, the pharmaceutical formulation containing a plurality of the coated IONRs is in a form suitable for intramuscular administration, intravenous administration, intraperitoneal administration, or subcutaneous administration, or a combination thereof.
Parenteral formulations containing the coated IONRs described herein are typically aqueous solutions which can contain excipients such as salts, carbohydrates and buffering agents (e.g., from about pH 6.5 to about pH 8.0, from about pH 6.5 to about pH 7.4, from about pH 6.5 to about pH 7.0, from about pH 7.0 to pH 8.0, or from about pH 7.0 to about pH 7.4), but, for some applications, they may be more suitably formulated as a sterile aqueous solution or as a dried form to be used in conjunction with a suitable vehicle such as sterile, pyrogen-free water.
The liquid formulations containing the coated IONRs for parenteral administration may be a solution, a suspension, or an emulsion.
The liquid pharmaceutically acceptable carrier forming the parenteral formulation containing the coated IONRs can include one or more physiologically compatible buffers, such as a phosphate buffers. One skilled in the art can readily determine a suitable saline content and pH for an aqueous carrier for administration (e.g., from about pH 6.5 to about pH 8.0, from about pH 6.5 to about pH 7.4, from about pH 6.5 to about pH 7.0, from about pH 7.0 to pH 8.0, or from about pH 7.0 to about pH 7.4).
Liquid formulations containing the coated IONRs for parenteral administration may include one or more suspending agents, such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, gum tragacanth, or lecithin. The liquid formulations may also include one or more preservatives, such as ethyl or n-propyl p-hydroxybenzoate.
Optionally, the liquid formulation containing the coated IONRs contains one or more solvents that are low toxicity organic (i.e., nonaqueous) class 3 residual solvents, such as ethanol, acetone, ethyl acetate, tetrahydrofuran, ethyl ether, and propanol, and a combination thereof. Any such solvents included in the liquid formulation should not detrimentally react with the one or more active agents present in the coated IONRs in the liquid formulation. Solvents such as freon, alcohol, glycol, polyglycol, or fatty acid, can also be included in the liquid formulation containing the coated IONRs as desired to increase the volatility of the solution or suspension.
Liquid formulations containing the coated IONRs for parenteral administration may also contain minor amounts of polymers, surfactants, or other pharmaceutically acceptable excipients known to those in the art. In this context, “minor amounts” means an amount that is sufficiently small to avoid adversely affecting uptake of the coated IONRs by the targeted cells, such as pituitary gonadotrophs.
The preparation of parenteral formulations containing the coated IONRs is typically under sterile conditions, for example, by lyophilisation, which can be accomplished using standard pharmaceutical techniques known to those skilled in the art.
Formulations for parenteral administration containing the coated IONRs may be formulated to provide immediate and/or modified release of the active agent. Modified release formulations include delayed, sustained, pulsed, controlled, targeted and programmed release formulations.
c. Amount of Coated IONRs
Pharmaceutical compositions typically contain an effective amount of the coated IONRs and one or more pharmaceutically acceptable carriers and/or excipients. As used herein, the term “effective amount” means any amount of the coated IONRs that is sufficient to achieve the desired therapeutic, prophylactic, and/or diagnostic effect on a biological sample or in a subject to which it is administered. Depending on the condition to be prevented, treated, or diagnosed, and/or the route of administration, such an effective amount of the coated IONRs can be between 0.01 to 1000 mg per kilogram body weight of the subject per day, between 0.1 and 500 mg, such as between 1 and 250 mg, for example about 5, 10, 20, 50, 100, 150, 200 or 250 mg, per kilogram body weight of the subject per day, which can be administered as a single daily dose, divided over one or more daily doses. The amount(s) of the coated IONRs administered, the route of administration, and the further treatment regimen can be determined by the treating clinician or testing technologies, depending on factors such as the age, gender and general condition of the subject, the nature and severity of the disease/symptoms being prevented, treated, or diagnosed, and/or the samples being tested.
In some forms, the total amount of the coated IONRs in the pharmaceutical composition can be in a range from about 0.001 wt % to about 20 wt %, from about 0.001 wt % to about 15 wt %, from about 0.001 wt % to about 10 wt %, from about 0.001 wt % to about 5 wt %, from about 0.001 wt % to about 2 wt %, from about 0.001 wt % to about 0.5 wt %, from about 0.001 wt % to about 0.2 wt %, from about 0.001 wt % to about 0.1 wt %, from about 0.01 wt % to about 20 wt %, from about 0.01 wt % to about 15 wt %, from about 0.01 wt % to about 10 wt %, from about 0.01 wt % to about 5 wt %, from about 0.01 wt % to about 2 wt %, from about 0.01 wt % to about 0.5 wt %, from about 0.01 wt % to about 0.2 wt %, from about 0.01 wt % to about 0.1 wt %, from about 0.1 wt % to about 20 wt %, from about 0.1 wt % to about 15 wt %, from about 0.1 wt % to about 10 wt %, from about 0.1 wt % to about 5 wt %, from about 0.1 wt % to about 2 wt %, from about 0.1 wt % to about 0.5 wt %, or from about 0.1 wt % to about 0.2 wt %. The term “total concentration of the coated IONRs in the pharmaceutical composition” refers to the sum of the weight of the coated IONRs in the composition relative to the weight of the composition.
In some forms, the pharmaceutical composition is in a unit dosage form, and can be suitably packaged, for example in a box, blister, vial, bottle, sachet, ampoule or in any other suitable single-dose or multi-dose holder or container (which can be properly labeled); optionally with one or more leaflets containing product information and/or instructions for use. Generally, such unit dosages can contain between 1 and 1000 mg, and usually between 5 and 500 mg, of the disclosed coated IONRs, e.g., about 10, 25, 50, 100, 200, 300 or 400 mg per unit dosage.
Kits and devices containing the coated IONR(s) are disclosed.
In some forms, the user is provided with a kit. The kit includes a device and the coated IONR(s). The device in the kit contains one or more defined region(s) or well(s), which are configured to contain a sample for in vitro applications, such as biomolecule (e.g., cell) separation or detection. The defined region(s) and well(s) of the device can have any suitable volume. For example, each of the defined region or well of the device independently has a volume of less than 100, 50, 25, 10, 5, 4, 3, 2 or 1 cubic centimeters. Optionally, the defined region or well of the device used in the methods is surrounded, lined, or fabricated using a material resistant to water, e.g., glass or plastic. In some forms, the device of the kit contains a plurality of defined regions or wells, e.g., more than or at least 5, 10, 20, 40, 80, 100, 200, or 400 defined regions or wells within one square foot area of the device, such as at least 80 wells within a microtiter plate.
Typically, in the kit, the coated IONR(s) are stored separately from the device prior to use, such as contained in a package or container separate from the device. In some forms, the coated IONR(s) are provided in the kit in the form of a pharmaceutical composition, such as in the form of a suspension in a buffered aqueous solution. Optionally, the kit further includes written instructions for performing a test using the device and coated IONRs.
When in use, the user can load the coated IONRs in one or more of the defined region(s) or well(s) and then mix a sample with the coated IONRs in the defined region(s) or well(s) of the device in the kit. Alternatively, the user can mix the coated IONRs with a sample and then load the coated IONRs-sample mixture in one or more of the defined region(s) or well(s) of the device in the kit.
In some forms, the user is provided with just a device containing one or more defined region(s) or well(s) and the coated IONRs. In these forms, the coated IONRs are contained in the defined region(s) or well(s) of the device. Optionally, the defined region or well or each defined region or well of the device is sealed to prevent leaking of the coated IONRs contained therein. The coated IONRs contained in the defined region(s) or well(s) of the device may be in solid form and/or liquid form. The defined region(s) or well(s) of the device can have any suitable volume such as any one of those described above. In some forms, the device is configured to contain a plurality of defined regions or wells, such as any one of the configurations described above.
Methods of making the coated IONRs that have strong magnetic properties are described. Coating iron oxide particles that have strong magnetic properties is a major limitation in the applications of such materials, because they tend to aggregate and form clusters in the coating process, which results in insufficient coating of these materials and thus poor aqueous stability (see, for example, Dynabeads® and magnetic beads supplied by StemCell Technology). To achieve sufficient coating of the iron oxide particles, currently available materials use iron oxide particles with relatively weak magnetic properties, leading to coated materials with low separation efficiency (see, for example, magnetic beads supplied by Miltenyi Biotec). The methods disclosed herein can form a sufficient coating on iron oxide nanorods that have strong magnetic properties, and making it possible to produce coated IONRs having both excellent stability in an aqueous medium (i.e., stay suspended in the aqueous medium for at least 1 hour, at room temperature) and superior magnetic property (i.e., a magnetic moment of at least 10 emu/g, induced using 1 T magnetizing field strength, at room temperature).
Generally, the method of making the coated IONRs includes: (i) mechanically mixing (such as by inverting, shaking, or stirring) a mixture comprising IONRs, a coating material, and a solvent for a time period in a range from about 12 hours to about 60 hours, from about 12 hours to about 48 hours, or from about 24 hours to about 48 hours, at a temperature in a range from 20° C. to about 40° C., such as about 25° C., to form a product comprising coated IONRs; and optionally (ii) during step (i), periodically sonicating the mixture for at least 1 min, at least 2 mins, at least 5 mins, at least 10 mins, at least 20 mins, at least 30 mins, or in a range from 1 min to 1 hour, from 1 min to 30 mins, from 1 min to 20 mins, from 1 min to 10 mins, or from 1 min to 5 mins, at room temperature. Step (ii) can be performed at a regular time interval or irregular time interval. For example, step (ii) is performed regularly every 30 mins, every hour, or every 2 hours during step (i), or step (ii) is performed irregularly with a time interval of 30 mins, 1 hour, and/or 2 hours. The IONRs being coated have a magnetic moment of at least 10 emu/g, at least 20 emu/g, at least 50 emu/g, or at least 80 emu/g, induced using 1 T magnetizing field strength, at room temperature.
The coating material in the mixture of step (i) can be any material suitable for coating IONRs, such as any one of the polymers described above. For example, the coating material is an amphiphilic polymer having Formula I or II described above.
The solvent in the mixture of step (i) is typically a polar organic solvent capable of dissolving the coating material, such as the amphiphilic polymer having Formula I or II described above. Examples of polar organic solvent suitable for use in the method include, but are not limited to, tetrahydrofuran, chloroform, DMSO, or DMF, or a combination thereof.
Generally, the concentration of the IONRs in the mixture of step (i) can be from about 0.001 mg Fe/mL to about 0.5 mg Fe/mL. The weight ratio between the iron oxide nanorods and the coating material in the mixture of step (i) can be from 500:1 to 5:1, from 100:1 to 5:1, from 50:1 to 5:1, or from 20:1 to 5:1.
Optionally, the disclosed method of making the coated IONRs further includes dispersing IONRs in the solvent to form a dispersion and sonicating the dispersion for a period in a range from about 1 min to about 10 mins, prior to step (i); dissolving the coating material in the solvent to form a solution and adding the solution into the dispersion to form the mixture, prior to step (i); subsequent to step (i), removing one or more of the solvent(s) in the mixture (such as chloroform) by a suitable method, such as by vaporization under vacuum; dialyzing the mixture against deionized water for a period in a range from 12 hour to 60 hours, from 24 hour to 60 hours, from 12 hour to 48 hours, from 12 hour to 24 hours, from 24 hour to 48 hours, from 12 hour to 36 hours, from 24 hour to 36 hours, subsequent to step (i), where the deionized water has a volume that is 2-10 times of the volume of the mixture, such as a volume that is about 4-time of the volume of the mixture; and/or collecting the coated IONRs using a magnet and filtering the collected coated IONRs through a filter, optionally wherein the filter has a size of 0.4 μm or less, subsequent to step (i) or dialysis against deionized water.
More specific coating conditions and an exemplary method of making the coated IONRs are described in the Examples below.
The disclosed coated IONRs have excellent aqueous stability and superior magnetic property, and thereby are suitable for use in a wide range of in vivo and in vitro biomedical applications, such as for use in purifying and detecting biological substances, such as proteins, peptides, DNAs, RNAs, cells, extracellular vesicles, and cytokines, in vivo or in vitro, delivering active agents, and imaging as contrast agents, in a subject in need thereof.
A. Separation and/or Detection
In some forms, the disclosed coated IONRs can be used for separating and/or detecting biological substances, such as cells, by employing IONRs which contain a targeting moiety specific for a moiety on the biological substances of interest, such as cells of interest. Typically, the target biological substances, such as target cells, in the sample contain a moiety to which the targeting moiety of the coated IONRs can bind, such that the coated IONRs bind with the target biological substances, such as the target cells, upon mixing. The method generally includes (i) mixing the coated IONRs (containing a target-specific targeting moiety) with a sample containing target biological substances (such as target cells) and non-target substances to form a sample mixture; (ii) exposing the sample mixture to a magnetic field; and (iii) separating the target biological substances from the non-target substances in the sample. Steps (i) and (ii) can be performed sequentially or concurrently. The sample is typically a biological sample of a mammal, such as whole blood, plasma, serum, saliva, nasal swab, mucus, sputum (processed or unprocessed), bronchial alveolar lavage (BAL), bronchial wash (BW), cerebrospinal fluid (CSF), or urine. Upon exposure to the magnetic field, the target biological substances in the mixture are restrained to the magnetic field, thereby allowing non-target substances to be removed from the sample mixture and separated from the target biological substances.
In some forms, the disclosed coated IONRs can be used for detecting the presence of target biological substance(s) in a sample; the method generally includes (i) mixing the disclosed coated IONRs (containing a target-specific targeting moiety) or a composition containing the coated IONRs (containing a target-specific targeting moiety) disclosed herein with the sample to form a sample mixture, such that the coated IONRs bind to target biological substances (such as target cells) in the sample mixture; (ii) exposing the sample mixture to a magnetic field; and (iii) detecting the presence of the target biological substances (such as target cells) in the sample mixture. Separated target biological substances (such as target cells or other disease-related biomarkers) can be detected by methods known in the art, such as fluorescence microscopy after immunofluorescence staining, HPLC, and/or mass spectroscopy. Steps (i) and (ii) can be performed sequentially or concurrently.
In some forms, the methods of separating and/or detecting the presence of target biological substances (such as target cells) described above are performed using a device containing a defined region or well. Typically, the defined region or well of the device has a volume of less than 100, 50, 25, 10, 5, 4, 3, 2 or 1 cubic centimeters. Optionally, the defined region or well of the device used in the methods is surrounded, lined, or fabricated using a material resistant to water, e.g., glass or plastic. In some forms, the device used in the methods described above can contain a plurality of defined regions or wells, e.g., more than or at least 5, 10, 20, 40, 80, 100, 200, or 400 defined regions or wells within one square foot area of the device, such as at least 80 wells within a microtiter plate. When such a device containing one or more defined region(s) or well(s) is used in the methods of separating and/or detecting the presence of target biological substances (such as target cells), the disclosed coated IONRs may be pre-loaded in the defined region or well of the device, or mixed with a sample and then loaded in the defined region or well of the device. When the disclosed coated IONRs are pre-loaded in the defined region or well of the device, a sample is added into the defined region or well and mixed with the coated IONRs and then exposed to a magnetic field capable of restricting the movement of target biological substances (such as target cells) that are bound to the coated IONRs contained in the defined region or well of the device for subsequent separation and/or detection. When the disclosed coated IONRs are supplied separately from the device, a sample is mixed with the coated IONRs and then the sample mixture is loaded into the defined region or well, followed by exposure to a magnetic field for separation and/or detection.
In some forms, the disclosed coated IONRs can be used as contrast agents for imaging; the method generally includes (i) administering a pharmaceutical composition containing the coated IONRs to a subject; and (ii) applying electromagnetic radiation to a target region of the subject. In step (ii), one or more sets of image data are obtained and optionally displayed on a device, such as a computer screen. Optionally, after the pharmaceutical composition containing the coated IONRs are administered to the subject, a predetermined time period lapses to allow for uptake of the coated IONRs to a target in the subject, such as cancer cells. The predetermined time for waiting for coated IONRs uptake can be varied depending upon the type of condition/disease and concentration of coated IONRs administered.
In some forms, the target region of the subject being imaged using the disclosed methods can be an organ in the chest and/or abdomen, such as the heart, liver, biliary tract, kidneys, spleen, bowel, pancreas, and adrenal glands; a pelvic organ, such as the bladder, a reproductive organ, such as the breast, uterus, and ovaries in females and the prostate gland in males; blood vessels; brain and brain stem; lymph nodes. In some forms, the disclosed method is to aid in the diagnoses or monitor treatment for conditions, such as tumors of the chest, abdomen or pelvis; diseases of the liver, such as cirrhosis, and abnormalities of the bile ducts and pancreas; inflammatory bowel disease such as Crohn's disease and ulcerative colitis; heart problems, such as congenital heart disease; malformations of the blood vessels and inflammation of the vessels (vasculitis); or a fetus in the womb of a pregnant woman.
The terms “image” and “imaging” refer to a variety of information outputs and associated techniques for gathering useful information from administered coated IONRs and are meant to include all types of external scanning mechanisms for localizing the coated IONRs administered to a subject in need thereof. For example, in one form of basic imaging, spectroscopy is employed as an imaging technique to derive a general determination as to the appearance of coated IONRs and/or the level of coated IONRs in a localized region, which is indicative of the presence of a malignant tumor in the body of a subject to whom the coated IONRs are administered. After such a determination, further imaging can be undertaken (either contemporaneously or after a predetermined time interval) to determine the precise characteristics, size, shape, type, etc. of the area/tissue. This further imaging can employ different and/or more-sensitive imaging devices than those initially employed on the localized areas of coated IONRs. These further imaging devices may or may not be particularly sensitive to the coated IONRs. Such devices include, but are not limited to MRIs, etc. as described herein.
In some forms, the disclosed coated IONRs are used as contrast agents for magnetic resonance imaging (MRI) or magnetic resonance angiography (MRA); the method includes the general steps (i) and (ii) described above and subsequently, a magnetic resonance signal image data set is obtained and optionally displayed on a device, where the magnetic resonance signal image data set is associated with distribution of the coated IONRs in the imaged region of the subject. Optionally, the imaging method disclosed herein further includes administering a second contrast agent to the subject, e.g., comprising Gd3+, prior to, concurrently, or subsequent to the administration of the pharmaceutical composition containing the coated IONRs.
In some forms, the disclosed coated IONRs can be used for delivering one or more active agents to a subject in need thereof; the method generally includes (i) administering a pharmaceutical formulation containing the coated IONRs to the subject, where the coated IONRs contain one or more active agents embedded in the coating thereof. The administration step can occur one or more times. Typically, the total amount of the one or more active agents that are embedded in the coatings of the IONRs, in the pharmaceutical formulation, is effective to prevent, treat, and/or ameliorate one or more symptoms of a given disease or disorder of the subject in need thereof. The phrase “total amount” with respect to the total amount off the one or more active agents that are embedded in the coatings of the IONRs refers to the sum of the weight of all of the active agent(s) embedded in the coatings of all of the coated IONRs in the pharmaceutical formulation.
Optionally, the method includes administering one or more additional active agents (such as one or more anticancer agents described above), not contained in the coated IONRs, to the subject prior to, concurrently with, or subsequent to administration of the pharmaceutical formulation containing the coated IONRs. These additional active agents not contained in the coated IONRs may be included in the pharmaceutical formulation containing the coated IONRs or in a separate pharmaceutical formulation that does not contain the coated IONRs.
Methods of administration of the pharmaceutical formulations containing the coated IONRs can be oral, i.e., administration to or by way of the mouth, to provide uptake through the GI tract; or parenteral, such as intravenous administration. In systemic circulation, the coated IONRs may accumulate in diseased site.
The pharmaceutical formulations can be administered in a single dose or in multiple doses. Certain factors may influence the dosage required to effectively prevent, treat, or ameliorate the symptoms of a disease or disorder, including, but not limited to, the severity of the disease or disorder, previous preventions, the general health and/or age of the subject, and other diseases present. It will also be appreciated that the effective dosage of the active agent(s) used for prevention or treatment may increase or decrease over the course of particular prevention or treatment. Changes in dosage may result and become apparent from the results of assays.
Preventing a disease or disorder or the symptoms of the disease or disorder includes administering a pharmaceutical formulation containing the coated IONRs to a subject at risk for or having a predisposition for one or more symptom caused by a disease or disorder to cause cessation of a particular symptom of the disease or disorder, a reduction or prevention of one or more symptoms of the disease or disorder, a reduction in the severity of the disease or disorder, the complete ablation of the disease or disorder, or stabilization or delay of the development or progression of the disease or disorder, or to have a combination of these effects.
1. Diseases or Disorders being Treated
The pharmaceutical formulations described herein can be administered to a subject to prevent or treat any disease or disorder or ameliorate one or more symptoms associated with a disease or disorder.
The subject or patient is an individual who is the target of treatment using the disclosed formulations containing a plurality of the coated IONRs containing one or more active agents embedded in the coatings thereof. The subject can be a vertebrate, for example, a mammal. Thus, the subject can be a human. The subjects can be symptomatic or asymptomatic. The term does not denote a particular age or sex. Thus, adult and newborn subjects, whether male or female, are intended to be covered. A subject can also include a control subject or a test subject.
Diseases or disorders that can be treated using the drug delivery methods described herein include, but are not limited to, diabetes; autoimmune disorders (e.g. Crohn's disease, chronic arthritis, multiple sclerosis, Sjogren's disease, Lupus erythematosus, psoriasis, Celiac disease, etc.); cancer (breast cancer (e.g., metastatic or locally advanced breast cancer), prostate cancer (e.g., hormone refractory prostate cancer), renal cell carcinoma, lung cancer (e.g., small cell lung cancer and non-small cell lung cancer (including adenocarcinoma, squamous cell carcinoma, bronchoalveolar carcinoma and large cell carcinoma)), pancreatic cancer, gastric cancer (e.g., gastroesophageal, upper gastric or lower gastric cancer), colorectal cancer, squamous cell cancer of the head and neck, ovarian cancer (e.g., advanced ovarian cancer, platinum-based agent resistant or relapsed ovarian cancer), lymphoma (e.g., Burkitt's, Hodgkin's or non-Hodgkin's lymphoma), leukemia (e.g., acute myeloid leukemia) and gastrointestinal cancer); pain; fungal infections; bacterial infections; inflammation; anxiety; etc.
The disclosed compositions and methods can be further understood through the following numbered paragraphs.
1. Coated iron oxide nanorods (IONRs) comprising:
2. The coated IONRs of paragraph 1, wherein the iron oxide core has a length in a range from about 20 nm to about 250 nm and a diameter in a range from about 2 nm to about 50 nm.
3. The coated IONRs of paragraph 1 or 2, wherein the coating comprises one or more amphiphilic polymers, wherein the amphiphilic polymer or each amphiphilic polymer has a structure of:
4. The coated IONRs of paragraph 3, wherein the amphiphilic polymer or each amphiphilic polymer has a structure of:
5. The coated IONRs of paragraph 1 or 2, wherein the coating comprises one or more PEG lipids, wherein the PEG lipid or each PEG lipid has a structure of:
6. The coated IONRs of paragraph 5, wherein L1 is a bond,
7. The coated IONRs of paragraph 5 or 6, wherein L1 is
8. The coated IONRs of any one of paragraphs 5-7, wherein R1 is
9. The coated IONRs of any one of paragraphs 5-8, wherein L2 is
10. The coated IONRs of any one of paragraphs 5-9, wherein L3 is
Z3 and Z4 are independently a bond or
n8 is an integer from 1 to 6.
11. The coated IONRs of any one of paragraphs 5-10, wherein L4 is hydrogen.
12. The coated IONRs of any one of paragraphs 5-10, wherein L4 is a bond,
Z5 and Z6 are independently a bond or
n9 is an integer from 1 to 6.
13. The coated IONRs of any one of paragraphs 5-12, wherein R2 and R3 are independently a C7-C30 or C7-C20 linear alkyl, C7-C30 or C7-C20 linear alkenyl, or C7-C30 or C7-C20 linear alkynyl, optionally substituted with one or more substituent, such as one or more —OH, —COOH, —COO-alkyl, —SH, —NH2, etc.
14. The coated IONRs of any one of paragraphs 5-13, wherein E1 is
n4 and n5 are independently an integer from 1 to 500, from 1 to 200, from 1 to 100, from 1 to 50, from 5 to 1000, from 5 to 500, from 5 to 200, from 5 to 100, from 5 to 50, from 10 to 1000, from 10 to 500, from 10 to 200, from 10 to 100, from 10 to 50, from 20 to 1000, from 20 to 500, from 20 to 200, or from 20 to 100, such as from 20 to 50.
15. The coated IONRs of any one of paragraphs 5-14, wherein the PEG lipid or each PEG lipid has a structure of:
16. The coated IONRs of any one of paragraphs 1-15, wherein the iron oxide core comprises Fe3O4 magnetite or a combination of Fe3O4 magnetite and FeO(OH) goethite.
17. The coated IONR of any one of paragraphs 1-16, having a hydrodynamic length in a range from about 50 nm to about 300 nm, from about 100 nm to about 300 nm, from about 50 nm to about 200 nm, from about 50 nm to about 150 nm, from about 150 nm to about 300 nm, from about 200 nm to about 300 nm, or from about 220 nm to about 300 nm, and a hydrodynamic width in a range from about 20 nm to about 150 nm, from about 20 nm to about 120 nm, from about 20 to about 100 nm, or from about 50 nm to about 150 nm.
18. The coated IONR of any one of paragraphs 1-17, having a zeta potential in a range from about −10 mV to about −100 mV.
19. The coated IONR of any one of paragraphs 1-18, is dispersed in an aqueous medium for at least 30 mins, at least 1 hour, at least 2 hours, at least 3 hours, or at least 4 hours, at room temperature.
20. The coated IONR of any one of paragraphs 1-19, having a separation efficiency of at least 80%, at least 85%, at least 90%, or at least 95%, within 1 min of magnet time.
21. The coated IONR of any one of paragraphs 3, 4, and 16-20, wherein the amphiphilic polymer or each amphiphilic polymer has a structure of:
22. The coated IONR of any one of paragraphs 3, 4, and 16-21, wherein L′ and/or L1 and L2 are independently a single bond or a linker selected from the group consisting of —NH—, —CH2—, —S—, —S—S—, —O—, —(C═O)O—, —O(C═O)—, and —(C═O)CH(CH3)—, or a combination thereof.
23. The coated IONR of any one of paragraphs 3, 4, and 16-22, wherein at least one T′ and/or at least one of T1 and T2 is, optionally each T′ and/or both T1 and T2 is/are independently a targeting moiety selected from the group consisting of an antibody, an antibody fragment, an antibody mimetic, an affibody, a nucleic acid, an oligonucleotide, an aptamer, a peptide, a steroid, histidine tag, NTA-Ni, and a tyrosine kinase inhibitor, or a combination thereof.
24. The coated IONR of any one of paragraphs 3, 4, and 16-23, wherein each L′ and/or both L1 and L2 is/are a single bond and each T′ and/or both T1 and T2 is/are hydrogen.
25. A pharmaceutical composition comprising one or more the coated IONR of any one of paragraphs 1-24 and a pharmaceutically acceptable carrier and/or excipient.
26. A kit comprising a device and the coated IONRs of any one of paragraphs 1-24, wherein the device comprises one or more defined region(s) or well(s).
27. A device comprising one or more defined region(s) or well(s) and the coated IONRs of any one of paragraphs 1-24, wherein each defined region or well contains one or more of the IONRs.
28. A method of producing coated iron oxide nanorods (IONRs), comprising:
29. The method of paragraph 28, wherein step (ii) is performed regularly every 30 mins, every hour, or every 2 hours during step (i), or wherein step (ii) is performed irregularly with a time interval of 30 mins, 1 hour, and/or 2 hours.
30. The method of paragraph 28 or 29, wherein the IONRs have a magnetic moment of at least 10 emu/g, at least 20 emu/g, at least 50 emu/g, or at least 80 emu/g, induced using 1 T magnetizing field strength, at room temperature.
31. The method of any one of paragraphs 28-30, wherein the coating material is an amphiphilic polymer, optionally wherein the amphiphilic polymer is a PEG lipid.
32. The method of any one of paragraphs 28-31, wherein the polymer has a structure of:
33. The method of any one of paragraphs 28-32, wherein the polymer has a structure of:
34. The method of any one of paragraphs 28-33, wherein the polymer has a structure of:
35. The method of any one of paragraphs 28-31, wherein the polymer is a PEG lipid, optionally wherein the PEG lipid has a structure of:
36. The method of any one of paragraphs 28-35, wherein the solvent is a polar organic solvent, optionally wherein the polar organic solvent is tetrahydrofuran, chloroform, DMSO, or DMF, or a combination thereof.
37. The method of any one of paragraphs 28-36, wherein the concentration of IONRs in the mixture is in a range from about 0.001 mg Fe/mL to about 0.5 mg Fe/mL.
38. The method of any one of paragraphs 28-37, wherein the weight ratio between the iron oxide nanorods and the coating material in the mixture is in a range from 500:1 to 5:1, from 100:1 to 5:1, from 50:1 to 5:1, or from 20:1 to 5:1.
39. The method of any one of paragraphs 28-38, further comprising dispersing IONRs in the solvent to form a dispersion and sonicating the dispersion for a period in a range from about 1 min to about 30 mins, prior to step (i).
40. The method of paragraph 39, further comprising dissolving the coating material in the solvent to form a solution and adding the solution into the dispersion to form the mixture, prior to step (i).
41. The method of any one of paragraphs 28-40, further comprising dialyzing the product comprising the coated IONRs against deionized water for a period in a range from 12 hour to 48 hours.
42. The method of any one of paragraphs 28-41, further comprising collecting the coated IONRs in the product using a magnet and filtering the collected coated IONRs through a filter, optionally wherein the filter has a size of 0.4 μm or less.
43. A method of separating target biological substances comprising:
44. A method of detecting target biological substances in a sample, comprising:
45. A method of imaging, comprising:
46. The method of paragraph 45, wherein in step (ii) one or more sets of image data are obtained and optionally displayed on a visual display.
47. A method of delivering one or more active agent to a subject in need thereof, comprising:
Two different protocols (Wan J, et al., J. Cryst. Growth. 2005, 276: 571-576; Arijit Mitra, et al., J. Phys. D: Appl. Phys. 2018, 51:085002) were used to synthesize IONRs (also referred to herein as iron oxide core), with modifications. The synthesized IONRs were coated using PEG-b-AGE. The preparation and structure of PEG-b-AGE are described in U.S. patent Ser. No. 10/393,736. It is noted that the coating protocol described in U.S. patent Ser. No. 10/393,736 cannot be used to produce PEG-b-AGE coated IONRs due to the strong magnetic property of the IONRs described herein. The details for IONRs synthesis and coating with PEG-b-AGE thereon are described below.
Benzene (8 mL) was laid above 40 ml distilled water in a Teflon liner of 60 ml capacity. Solvents were sonicated for 20 min, followed by purging with argon gas for 20 min. Then 1.0 mmol FeSO4·7H2O and 2.0 mmol FeCl3 were added to be dissolved in the purged distilled water at room temperature. After iron salts dissolved, ethylenediamine (5 mL) was added. The liner was sealed in a stainless-steel autoclave and maintained at 120° C. for 20 h, then cooled to room temperature. The resulting black solid products were filtered off, washed with distilled water and absolute ethanol several times, and dried in oven to yield products in solid form.
Synthesis of precursor: 3 g FeCl3·6H2O was mixed with varying amounts of PEI (0, 0.1, or 0.2 mL) in 20 mL deionized H2O under magnetic stirring. The mixture was placed in autoclave and reacted at 120° C. for 4 h.
Synthesis of IONRs: β-FeOOH (100 mg) precursor was dispersed in 10 mL of oleylamine and heated to 120° C. for 15 min, followed by heating to 220° C. for 4-5 h. The reaction was carried out in a strict oxygen-free atmosphere under a constant flow of nitrogen. Oleylamine functions as a solvent, reducing agent, and surface functionalizing agent.
IONRs were dispersed in a polar organic solvent, such as tetrahydrofuran (THF), chloroform, DMSO, or DMF, or a mixture thereof, and sonicated for one min. PEG-b-AGE polymer was dissolved in the same solvent and added to the IONRs solution. The final concentration of IONRs is 0.1-0.2 mg Fe/mL. The weight ratio between PEG-b-AGE polymer and IONRs is 10:1. The mixture was periodically sonicated for about one min during the mechanical stirring process at room temperature for 48 hours. Afterwards, the mixture was added to deionized (DI) water with 4 times volume and dialyzed against DI water. The resultant IONRs were collected magnetically and filtered through 0.4 μm filter to remove large aggregates.
The IONRs were characterized using TEM. The TEM image (scale bar 100 nm) showed that the IONRs synthesized following protocols 1 and 2 have an average length of about 200 nm (protocol 1;
The PEG-b-AGE coated INORs (produced following protocols 1 and 2) were characterized by TEM, dynamic light scattering (DLS), and zeta potential. Specifically, hydrodynamic dimensions of the coated IONRs can be measured using dynamic light scattering; and both the hydrodynamic dimensions and zeta potential of the coated IONRs can be measured using a Malvern Zetasizer instrument. The TEM image and DLS data showed that the PEG-b-AGE coated IONRs (which have a core length of about 200 nm) have an aspect ratio (length/width) of about 6 to 9 (
Particles self-precipitation is an issue that limits use of magnetic particles in biomedical applications. The amount of magnetic materials dispersed in solution was quantified based on the UV absorbance at their corresponding peaks. Particles were vortexed to ensure full dispersion before testing. Absorbance of the fully dispersed solution was set as 100% for data normalization. As shown in
Further, the efficiency of PEG-b-AGE coated IONRs pulled out by a magnet from solution was compared with commercially available magnetic beads (i.e., magnetic beads available from Militenyi Biotec (Lot #: 5221009844), Dynabead® (Lot #: 00747022), and magnetic beads available from StemCell Technology (Lot #: 1000096771)). The amount of magnet material dispersed in solution was quantified based on the UV absorbance at their corresponding peaks. Results were normalized with the content before exposure to a magnet set as 100%. As shown in
Although Dynabeads® and StemCell Technology's magnetic beads showed a similar efficiency of magnet separation compared to the PEG-b-AGE coated IONRs, they showed a much shorter stable dispersion period than PEG-b-AGE coated IONRs (
IONRs were synthesized according to the Synthesis Protocol 2 described in Example 1 above. Iron oxide nanorods (IONR) were initially dispersed in chloroform and mixed with an exemplary coating polymer (structure shown below) in chloroform. DMSO was then added to the mixture. The mixture was incubated on a shaker at room temperature. Afterwards, chloroform was removed by vaporization under vacuum. Deionized water was then added to the residue, and DMSO was then removed by dialysis.
The coated INORs were characterized by TEM and dynamic light scattering (DLS). TEM images of polymer coated IONRs with 50 nm (
Particles self-precipitation was measured to assess the stability of the coated IONRs. The amount of magnetic materials dispersed in solution was quantified based on the UV absorbance at their corresponding peaks. Particles were vortexed to ensure full dispersion before testing. Absorbance of the fully dispersed solution was set as 100% for data normalization. As shown in
Further, the efficiency of the exemplary polymer coated IONRs pulled out by a magnet from solution was tested. The efficiency was quantified based on the UV absorbance of coated IONRs at corresponding peaks. Results were normalized with the content before exposure to a magnet as 100%. As shown in
This application claims the benefit of and priority to U.S. Provisional Application No. 63/500,389 filed May 5, 2023, the entire content of which is incorporated herein by reference for all purpose in its entirety.
This invention was made with government support under Grant No. AG078718 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 63500389 | May 2023 | US |
