CN116669773A - Method for functionalizing nanoparticles - Google Patents
Method for functionalizing nanoparticles Download PDFInfo
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- CN116669773A CN116669773A CN202180073510.XA CN202180073510A CN116669773A CN 116669773 A CN116669773 A CN 116669773A CN 202180073510 A CN202180073510 A CN 202180073510A CN 116669773 A CN116669773 A CN 116669773A
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- nanoparticle
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Abstract
The present disclosure relates to functionalized nanoparticles, e.g., for conjugation to targeting ligands and/or payload moieties, such as methods for producing Nanoparticle Drug Conjugates (NDCs).
Description
RELATED APPLICATIONS
The present application claims U.S. provisional application No. 63/105,995 filed on day 27 of 10 in 2020, U.S. provisional application No. 63/116,393 filed on day 11 and 20 in 2020, U.S. provisional application No. 63/117,110 filed on day 23 in 11 in 2020, U.S. provisional application No. 63/155,043 filed on day 3 in 2021, U.S. provisional application No. 63/222,181 filed on day 15 in 7 in 2021, U.S. provisional application No. 63/242,201 filed on day 9 in 2021, and U.S. provisional application No. 63/254,837 filed on day 12 in 10 in 2021, the contents of which are all incorporated herein by reference.
Background
Nanoparticle Drug Conjugates (NDCs) offer the possibility to address numerous drawbacks of other drug carrier platforms, such as Antibody Drug Conjugates (ADCs). For example, NDCs with ultra-small dimensions (e.g., 20nm or less in diameter) can cross a disrupted blood brain barrier and deliver therapeutic drugs to brain tumors, which cannot be achieved using other platforms (e.g., ADCs). Ultra-small nanoparticles can exhibit deep tumor penetration and provide uniform delivery of therapeutic molecules to tumors, while other drug carrier platforms (e.g., ADCs) have limited tumor penetration due to slow intratumoral diffusion. NDCs can also carry many more drug molecules than traditional drug delivery platforms (e.g., ADCs), which allow NDCs to deliver relatively large amounts of drugs to cancer cells. This is particularly useful in targeting cancer cells with low receptor expression that complicates the delivery of sufficient amounts of drug. Another advantage of NDC is that nanoparticles can be coated with an organic polymer layer, e.g., coating the nanoparticle surface with a layer of PEG groups, which can prevent serum proteins from adsorbing onto the nanoparticles in a physiological environment (e.g., in a subject), and can promote efficient urine excretion and reduce aggregation of the nanoparticles (see, e.g., burns et al, "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", nano Letters (2009) 9 (1): 442-448).
However, there are significant obstacles in the development and manufacture of NDCs. For example, it is very challenging to create NDCs that meet stringent standards in terms of production control, stability, drug release, safety, and efficacy required for clinical transformations. In particular, creating a connection between a conjugated molecule (e.g., a drug molecule and/or a targeting ligand) and a nanoparticle carrier that meets these criteria is very difficult.
Thus, the need for a method of functionalizing nanoparticles and allowing conjugation of molecules to the nanoparticles has not been met.
Disclosure of Invention
The present disclosure features methods of functionalizing nanoparticles. The method may include preparing a nanoparticle (e.g., a targeting ligand, e.g., a cancer targeting ligand, e.g., a folate receptor targeting ligand, e.g., folic acid) suitable for conjugating a compound to a nanoparticle surface, and a therapeutic agent, e.g., a cytotoxic compound, e.g., isatecan (exec)) to form a Nanoparticle Drug Conjugate (NDC). The resulting NDCs exhibit both highly stable attachment of the conjugate molecule to the nanoparticle and provide efficient drug release in targeted biological systems (e.g., cancer cells).
The synthetic methods disclosed herein may involve a series of reactions that may introduce a series of different reactive groups (sometimes referred to herein as functional groups) or compounds (e.g., payload moieties or targeting ligands) to the nanoparticle surface. These reactions may include, among others (e.g., other reactions described herein), silane condensation reactions, diels-Alder reactions, and "click chemistry", 2+3 or 2+4 cycloaddition reactions, and the like. The method can be used to functionalize ultra-small nanoparticles, including nanoparticles coated with organic polymers and/or silica nanoparticles (e.g., ultra-small pegylated silica nanoparticles, e.g., C' Dot).
The method of functionalizing a nanoparticle may include contacting a nanoparticle with a first bifunctional precursor, e.g., a bifunctional precursor comprising a silane moiety and another reactive group (e.g., a diene, an amine, a thiol, a hydroxyl, an azide, an alkene, or an alkyne), wherein the nanoparticle comprises a surface that is reactive with a silane (e.g., a silica surface), and wherein the contacting is performed under conditions suitable for the reaction between the silane moiety and the surface of the nanoparticle (e.g., conditions described herein for silane condensation), thereby forming a covalent bond between the silane moiety and the surface of the nanoparticle, and providing a nanoparticle functionalized with the reactive group (e.g., a diene, an amine, a thiol, an azide, an alkene, or an alkyne). It should be appreciated that the method may be used to provide nanoparticles comprising a plurality of reactive groups.
The contacting of the nanoparticle with the first bifunctional precursor may involve inserting the bifunctional precursor into interstitial spaces between organic polymer molecules (e.g., PEG chains) attached to the nanoparticle, e.g., when using a nanoparticle comprising a surface coated with PEG. Thus, this approach overcomes difficulties in conventional methods of nanoparticle functionalization that are not effective in delivering precursor molecules to the surface of nanoparticles comprising an organic polymer layer (e.g., a PEG molecular layer) for functionalization.
The methods disclosed herein can further include contacting the nanoparticle functionalized with a reactive group (e.g., a nanoparticle functionalized with a diene, amine, thiol, hydroxyl, azide, alkene, or alkyne) with a second bifunctional precursor, wherein the second bifunctional precursor comprises a group that reacts with the reactive group on the nanoparticle, and wherein the second bifunctional precursor comprises another functional group (e.g., an alkyne moiety (e.g., dibenzoazacyclooctyne (DBCO)), a diene, amine, thiol, hydroxyl, alkene, or azide). For example, the second bifunctional precursor may comprise a dienophile (e.g., maleimide) that reacts with a reactive group (e.g., diene) on the nanoparticle, and wherein the second bifunctional precursor may also comprise an alkyne moiety (e.g., DBCO). The contacting can be performed under conditions suitable for the reaction between the functionalized nanoparticle and the second bifunctional precursor (e.g., the reaction conditions disclosed herein) to covalently bind the second bifunctional precursor to the nanoparticle and provide the nanoparticle functionalized with another functional group (e.g., an alkyne moiety, such as DBCO). It should be appreciated that the method may be used to provide nanoparticles functionalized with multiple additional functional groups (e.g., multiple DBCO moieties).
The method can further include contacting a nanoparticle functionalized with additional functional groups (e.g., a DBCO functionalized nanoparticle) with a compound comprising groups reactive with the additional functional groups on the nanoparticle. For example, the compound may comprise an alkyne reactive group (e.g., azide, diene, nitrone, or nitrile oxide) that is suitable for reacting with an alkyne group of an alkyne-functionalized nanoparticle. The contacting can be performed under conditions suitable for the reaction between the alkyne moiety and the alkyne-reactive group (e.g., the reaction conditions described herein, e.g., click chemistry conditions or other cycloaddition reaction (e.g., 3+2 cycloaddition reaction or 4+2 cycloaddition reaction conditions)), thereby forming the nanoparticle functionalized with the compound. The compound comprising a group that reacts with a functional group on the nanoparticle may comprise a payload moiety (e.g., a cytotoxic drug as disclosed herein, e.g., isatecan) or a targeting ligand (e.g., a folate receptor targeting ligand, e.g., folic acid). The method can be used to covalently attach a variety of compounds to the nanoparticle surface. For example, the method can introduce a plurality of compounds comprising groups reactive with the functionalized nanoparticle, or introduce a plurality of different compounds, each compound each comprising a group reactive with the functionalized nanoparticle (e.g., a plurality of targeting ligands, a plurality of payload moieties, or a combination thereof), and covalently attach the compound to the nanoparticle.
It will be appreciated that each step of the method may be used to introduce a plurality of functional groups into the nanoparticle. For example, the nanoparticle may be contacted with a plurality of first bifunctional precursors comprising a silane group and a reactive group, providing a nanoparticle functionalized with a plurality of reactive groups (e.g., a plurality of diene moieties). The nanoparticle functionalized with a plurality of reactive groups (e.g., a plurality of diene moieties) may be contacted with a plurality of second bifunctional precursors, wherein the second bifunctional precursors comprise groups that react with the reactive groups on the nanoparticle, and wherein the second bifunctional precursors comprise additional functional groups (e.g., alkynes, e.g., DBCO), thereby providing a nanoparticle functionalized with a plurality of functional groups (e.g., a plurality of DBCO moieties) from the second bifunctional precursors. Nanoparticles functionalized with a plurality of functional groups from the second bifunctional precursor may then be contacted with a plurality of compounds comprising groups reactive with the functional groups on the nanoparticles to provide nanoparticles comprising the plurality of compounds. Nanoparticles functionalized with a plurality of functional groups from the second bifunctional precursor may be contacted with a first plurality of compounds comprising groups reactive with the functional groups on the nanoparticles (e.g., targeting ligands comprising azides) and subsequently contacted with a second plurality of compounds comprising groups reactive with the functional groups on the nanoparticles (e.g., payload-linker conjugates comprising azides), wherein the first and second plurality of compounds comprise structurally different compounds, thereby providing nanoparticles comprising two different pluralities of compounds (e.g., targeting ligands and payload moieties).
For example, the method can include forming a nanoparticle functionalized with a plurality of diene moieties (e.g., cyclopentadiene moieties), e.g., by contacting the nanoparticle with a plurality of first bifunctional precursors comprising a silane moiety and a diene moiety under conditions sufficient to react the silane moiety with the surface of the nanoparticle. The method can include forming a nanoparticle functionalized with a plurality of alkyne moieties (e.g., DBCO moieties), for example, by contacting a nanoparticle comprising a plurality of diene moieties (e.g., cyclopentadiene moieties) with a plurality of second bifunctional precursors comprising a dienophile (e.g., maleimide) and an alkyne moiety (e.g., DBCO) under conditions sufficient to react the diene moieties and the dienophile. The method may further comprise: (a) Reacting a first portion of the plurality of alkyne moieties on the nanoparticle with a first compound comprising alkyne reactive groups (under conditions sufficient for reaction between alkyne moieties and alkyne reactive groups to occur); (b) A second portion of the plurality of alkyne moieties on the nanoparticle is reacted with a second compound comprising alkyne reactive groups (under conditions sufficient for reaction between alkyne moieties and alkyne reactive groups to occur) to form the nanoparticle functionalized with the plurality of first compounds and the plurality of second compounds, wherein the first compounds and the second compounds are chemically different. For example, the (a) first compound may be a compound of formula (D) disclosed herein (e.g., a compound of formula (D-1)); and the second compound may be a compound of formula (E) (e.g., a compound of formula (E-1)) as disclosed herein. Alternatively, the first compound may be a compound of formula (E) (e.g., a compound of formula (E-1)) disclosed herein, and the second compound is a compound of formula (D) (e.g., a compound of formula (D-1)) disclosed herein.
The present disclosure also relates to a method of functionalizing silica nanoparticles comprising: (i) Contacting a silica nanoparticle with a first bifunctional precursor, wherein the first bifunctional precursor comprises a silane moiety and a cyclopentadiene moiety, wherein the silica nanoparticle comprises a surface (e.g., a silica surface) that reacts with the silane moiety, and wherein the contacting is performed under conditions suitable for a reaction between the silane moiety and the silica nanoparticle surface, thereby forming a covalent bond between the silane moiety and the silica nanoparticle surface, and providing a nanoparticle functionalized with a cyclopentadiene moiety; (ii) Contacting a nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor, wherein the second bifunctional precursor comprises an alkyne moiety (e.g., DBCO) and a dienophile (e.g., maleimide), wherein the contacting is performed under conditions suitable for a reaction between the dienophile and the cyclopentadiene moiety, thereby reacting the diene moiety of the nanoparticle with the second bifunctional precursor and providing a nanoparticle functionalized with an alkyne moiety; and (iii) contacting the alkyne moiety with a compound comprising an azide moiety under conditions suitable for reaction between the alkyne moiety and the azide moiety (e.g., click chemistry conditions), thereby reacting the alkyne moiety of the nanoparticle with the azide moiety and providing a nanoparticle functionalized with the compound (e.g., a compound comprising a payload or a targeting ligand).
The present disclosure also relates to a method of functionalizing silica nanoparticles comprising: (i) Contacting the silica nanoparticle with a first bifunctional precursor comprising the structure of formula (a), thereby providing a nanoparticle functionalized with a cyclopentadiene moiety; (ii) Contacting the nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor comprising the structure of formula (B), thereby providing a nanoparticle functionalized with an alkyne moiety; and (iii) contacting the alkyne moiety with a compound of formula (D) or formula (E) or both, thereby providing a nanoparticle functionalized with a targeting ligand, a payload moiety, or both.
The first bifunctional precursor may comprise a structure of formula (A-1) provided herein. The second dual-function precursor may comprise a structure of formula (B-1) provided herein. The compound of formula (D) may comprise the structure of formula (D-1) provided herein. The compound of formula (E) may comprise the structure of formula (E-1) provided herein.
The methods disclosed herein can provide nanoparticles comprising a compound of formula (NP-2) provided herein. The average ratio of nanoparticles to (NP-2) may be about 1:1 to about 1:50 (each nanoparticle comprises an average of 1-50 units of NP-2), for example, about 1:40, about 1:30, about 1:25, about 1:20, about 1:15, about 1:14, about 1:13, about 1:12, about 1:11, or about 1:10.
The method may provide nanoparticles comprising a compound of formula (NP-3) as provided herein. The average ratio of nanoparticles to (NP-3) may be about 1:1 to about 1:80 (each nanoparticle comprises an average of 1-80 units of NP-3), for example, about 1:60, about 1:40, about 1:30, about 1:28, about 1:26, about 1:25, about 1:24, about 1:23, about 1:22, about 1:21, about 1:20, about 1:19, or about 1:18.
The methods can provide nanoparticles comprising a compound of formula (NP-2) and a compound of formula (NP-3), the structure of which is provided herein, wherein the ratio of (NP-3): (NP-2) is about 1:20:10, 1:20:11, 1:20:12, 1:20:13, 1:20:14, 1:20:15, 1:21:10, 1:21:11, 1:21:12, 1:21:13, 1:21:14, 1:21:15, 1:22:10, 1:22:11, 1:22:12, 1:22:13, 1:22:14, 1:22:15, 1:23:10, 1:23:11, 1:23:12, 1:23:13, 1:23:14, 1:23:15, 1:24:10, 1:24:11, 1:24:12, 1:24:13, 1:24:14, 1:15, 1:25:25:25:25:25:25:25:25:11, or 1:25:15).
An advantage of the methods disclosed herein is that stable functionalized nanoparticles are produced using relatively stable precursors (e.g., diene-silane precursors). For example, functionalized nanoparticles (e.g., NDCs) that can be produced using the methods disclosed herein can avoid premature or unwanted cleavage, which can occur in functionalized nanoparticles (e.g., NDCs) produced using other methods or using other precursors. For example, other methods of functionalizing nanoparticles use precursors that produce nanoparticles with reactive moieties on the nanoparticle surface that can promote unwanted reactivity, possibly resulting in, for example, premature release of the payload or unwanted release of the targeting ligand. In addition, other methods of nanoparticle functionalization use precursors that are unstable and self-destruct during the reaction, resulting in unwanted aggregation. Aggregates can be difficult to separate from functionalized nanoparticles.
In contrast, the methods disclosed herein can use relatively stable precursors, and the resulting functionalized nanoparticles (e.g., NDCs) are stable and of high purity. For example, functionalized nanoparticles of the present disclosure can be prepared with a diene-silane precursor (e.g., a cyclopentadiene-silane precursor) to provide nanoparticles functionalized with one or more diene groups. The dienyl group may then be reacted with a second precursor, e.g., a dienophile-containing precursor (e.g., a PEG-maleimide derivative, e.g., DBCO-PEG-maleimide), resulting in the formation of a stable ring adduct. The resulting functionalized nanoparticles comprising the cycloadduct can optionally be reacted with one or more subsequent precursors (e.g., targeting ligand precursors and/or payload-linker conjugate precursors described herein) to further functionalize the nanoparticle. The diene-silane precursor and the resulting cycloadduct do not exhibit the undesirable characteristics present in other functionalized nanoparticles or precursors thereof. For example, functionalized nanoparticles (e.g., NDCs) prepared using the methods disclosed herein have higher serum stability and can be produced in high yields and purity (e.g., free of aggregated precursors). Furthermore, since this approach is highly modular, any desired proportion of payload, targeting ligand or other incorporation of nanoparticles can be incorporated. Examples of nanoparticles prepared using these methods and their benefits are provided in the examples.
An exemplary sequence of functionalized nanoparticles is depicted in scheme 1.
Scheme 1 modifies an exemplary sequence of nanoparticles (C' Dot).
Drawings
Fig. 1 illustrates the structure of an exemplary Nanoparticle Drug Conjugate (NDC) that can be prepared using the methods disclosed herein.
Fig. 2 is a graph comparing the efficiency between different methods of functionalizing nanoparticles with DBCO moieties.
FIGS. 3A-3D are RP-HPLC and SEC chromatograms. FIG. 3A provides RP-HPLC chromatograms of DBCO functionalized nanoparticles prepared using amine-based bifunctional precursors before and after 24 hours incubation in PBS. FIG. 3B provides RP-HPLC chromatograms of DBCO functionalized nanoparticles prepared using diene-based bifunctional precursors before and after 24 hours incubation in PBS. Fig. 3C provides SEC chromatograms of DBCO functionalized nanoparticles prepared using amine-based bifunctional precursors. Fig. 3D provides SEC chromatograms of DBCO functionalized nanoparticles prepared using diene-based bifunctional precursors.
FIG. 4 is a spectral diagram from Fluorescence Correlation Spectroscopy (FCS) of an exemplary NDC prepared using the methods disclosed herein, demonstrating average hydrodynamic diameters.
Fig. 5 shows overlapping UV-Vis spectra of exemplary functionalized nanoparticles at different stages of functionalization, demonstrating the presence and average number of characteristic absorption peaks that can be used to verify the presence of each conjugate (e.g., targeting ligand, such as Folic Acid (FA), or payload, such as isatecan) in each particle.
Fig. 6A-6B provide RP-HPLC chromatograms (fig. 6A) and SEC chromatograms (fig. 6B) at two wavelengths of an exemplary NDC prepared using the methods disclosed herein.
Fig. 7 is a graph providing the amount of drug released over time (isatecan) by an exemplary NDC when incubated with cathepsin-B at 37 ℃. The inset provides overlapping UV-Vis spectra recorded at each time point.
Fig. 8A-8B are graphs illustrating serum stability of exemplary NDCs prepared using the methods disclosed herein. Fig. 8A compares the stability of NDC produced using a diene-based bifunctional precursor and NDC produced using an amine-based bifunctional precursor in 10% human serum at 37 ℃ over 7 days. Fig. 8B compares the stability of NDC produced using a diene-based bifunctional precursor and NDC produced using an amine-based bifunctional precursor in 10% mouse serum at 37 ℃ over 7 days.
Detailed Description
Selected chemical definition
As used herein, the term "alkyl" refers to a group that may contain from 1 to 18 carbon atoms, for example, from 1 to about 12Monovalent aliphatic hydrocarbon radicals of 1 to about 6 carbon atoms ("C 1-18 Alkyl "). The alkyl group may be a straight chain, branched, monocyclic or polycyclic moiety or a combination thereof. Examples of alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, norbornyl, and the like. Each instance of alkyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkyl") or substituted ("substituted alkyl") with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent).
As used herein, the term "alkenyl" refers to a monovalent straight or branched hydrocarbon radical having 2 to 18 carbon atoms, one or more carbon-carbon double bonds, and no triplets ("C 2-18 Alkenyl "). Alkenyl groups may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. One or more of the carbon-carbon double bonds may be internal (e.g., in 2-butenyl) or terminal (e.g., in 1-butenyl). Examples of alkenyl groups include vinyl, 1-propenyl, 2-propenyl, 1-butenyl, 2-butenyl, butadienyl, pentenyl, pentadienyl, hexenyl, heptenyl, octenyl, xin San alkenyl and the like. Each instance of alkenyl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkenyl") or substituted ("substituted alkenyl") with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent).
As used herein, the term "alkynyl" refers to a monovalent straight or branched hydrocarbon radical ("C") having 2 to 18 carbon atoms and one or more carbon-carbon triple bonds 2-18 Alkynyl "). Alkynyl groups may have 2 to 8 carbon atoms, 2 to 6 carbon atoms, 2 to 5 carbon atoms, 2 to 4 carbon atoms, or 2 to 3 carbon atoms. The one or more carbon-carbon triple bonds may be internal (e.g., in 2-butynyl) or terminal (e.g., in 1-butynyl). Examples of alkynyl groups include ethynyl, 1-propynyl, 2-propynyl, 1-butynyl, 2-butynyl and the like. Each instance of alkynyl can independently be optionally substituted, i.e., unsubstituted (an "unsubstituted alkyne) A group ") or substituted with one or more substituents (e.g., 1 to 5 substituents, 1 to 3 substituents, or 1 substituent) (" substituted alkynyl ").
As used herein, the term "heteroalkyl" refers to a non-cyclic, stable straight or branched chain, or a combination thereof, comprising at least one carbon atom and at least one heteroatom selected from the group consisting of: o, N, P, si, and S, and wherein the nitrogen and sulfur atoms can optionally be oxidized, and the nitrogen heteroatom can optionally be quaternized. The heteroatoms O, N, P, S and Si can be located anywhere in the heteroalkyl group.
Unless otherwise indicated, the terms "alkylene", "alkenylene", "alkynylene" or "heteroalkylene", alone or as part of another substituent, refer to divalent groups derived from an alkyl, alkenyl, alkynyl or heteroalkyl group, respectively. Unless otherwise indicated, the term "alkenylene" by itself or as part of another substituent refers to a divalent group derived from an olefin. Alkylene, alkenylene, alkynylene or heteroalkylene may be described as, for example, C 1-6 Alkylene, C 1-6 Alkenylene radicals, C 1-6 Meta-alkynylene or C 1-6 A meta heteroalkylene, where the term "meta" refers to a non-hydrogen atom within the moiety. In the case of heteroalkylene groups, the heteroatom can also occupy either or both of the chain ends (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, and the like). Furthermore, the writing direction of the chemical formula of the linking group does not imply the direction of the linking group for alkylene and heteroalkylene linking groups. For example, -C (O) 2 R' -may represent-C (O) 2 R '-and-R' C (O) 2 -. Each instance of alkylene, alkenylene, alkynylene, or heteroalkylene may independently be optionally substituted, i.e., unsubstituted ("unsubstituted alkylene") or substituted ("substituted heteroalkylene").
As used herein, the terms "substituted alkyl", "substituted alkenyl", "substituted alkynyl", "substituted heteroalkyl", "substituted heteroalkenyl", "substituted heteroalkynyl", "substituted cycloalkyl", "substituted heterocyclyl", "substituted aryl" and "substituted heteroaryl" refer to alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl, cycloalkyl, heterocyclyl, aryl and heteroaryl moieties having substituents that have one or more hydrogen atoms in the moiety substituted on one or more carbons or heteroatoms, respectively. Such substituents may include, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylic acid, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthio carbonyl, alkoxy, phosphate, phosphono, phosphinoyl, amino (which includes alkylamino, dialkylamino, arylamino, diarylamino, and alkylarylamino), amido (which includes alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfinylamino, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety. Cycloalkyl groups may be further substituted, for example, with the substituents described above.
As used herein, the term "alkoxy" refers to a group of formula-O-alkyl. The term "alkyloxy" or "alkoxy" includes substituted and unsubstituted alkyl, alkenyl, and alkynyl groups covalently linked to an oxygen atom. Examples of alkyl or alkoxy radicals include, but are not limited to, methoxy, ethoxy, isopropoxy, propoxy, butoxy and pentoxy. Examples of substituted alkoxy groups include haloalkoxy groups. Alkoxy groups may be substituted with groups such as alkenyl, alkynyl, halogen, hydroxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylic acid, alkylcarbonyl, arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl, alkylthio carbonyl, alkoxy, phosphate, phosphono, phosphinoyl, amino (which include alkylamino, dialkylamino, arylamino, diarylamino, and alkylaryl amino), amido (which include alkylcarbonylamino, arylcarbonylamino, carbamoyl, and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxylate, sulfate, alkylsulfinyl, sulfonic acid, sulfamoyl, sulfinylamino, nitro, trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or aromatic or heteroaromatic moieties. Examples of halogen substituted alkoxy groups include, but are not limited to, fluoromethoxy, difluoromethoxy, trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.
As used herein, the term "aryl" refers to a stable aromatic ring system, which may be monocyclic or polycyclic, wherein all ring atoms are carbon, and may be substituted or unsubstituted. The aromatic ring system may have, for example, 3 to 7 ring atoms. Examples include phenyl, benzyl, naphthyl, anthracenyl, and the like. Each instance of aryl may independently be optionally substituted, i.e., unsubstituted ("unsubstituted aryl") or substituted by one or more substituents ("substituted aryl").
As used herein, the term "heteroaryl" refers to an aryl group that includes one or more ring heteroatoms. For example, heteroaryl groups may include stable 5-, 6-, or 7-membered monocyclic or 7-, 8-, or 9-membered bicyclic aromatic heterocycles composed of carbon atoms and one or more heteroatoms independently selected from the group consisting of: nitrogen, oxygen and sulfur. The nitrogen atom may be substituted or unsubstituted (e.g., N or NR 4 Wherein R is 4 Is H or other substituent as defined). Examples of heteroaryl groups include pyrrole, furan, indole, thiophene, thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole, oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and the like.
As used herein, the terms "cycloalkylene", "heterocyclylene", "arylene" and "heteroarylene", alone or as part of another substituent, represent divalent groups derived from cycloalkyl, heterocyclyl, aryl and heteroaryl, respectively. Each instance of cycloalkylene, heterocyclylene, arylene, or heteroarylene may be independently optionally substituted, i.e., unsubstituted ("unsubstituted arylene") or substituted with one or more substituents ("substituted heteroarylene").
As used herein, the term "cycloalkyl" is intended to include non-aromatic cyclic hydrocarbon rings, for example, hydrocarbon rings having three to eight carbon atoms in their ring structure. Cycloalkyl groups may include cyclobutyl, cyclopropyl, cyclopentyl, cyclohexyl, and the like. Cycloalkyl groups may be monocyclic ("monocyclic cycloalkyl") or contain fused, bridged or spiro ring systems, for example bicyclic systems ("bicyclic cycloalkyl"), and may be saturated or may be partially unsaturated. "cycloalkyl" also includes ring systems in which a cycloalkyl ring (as defined above) is fused to one or more aryl groups, wherein the attachment point is on the cycloalkyl ring, and in which case the number of carbons continues to represent the number of carbons in the cycloalkyl ring system. Each instance of cycloalkyl can independently be optionally substituted, i.e., unsubstituted ("unsubstituted cycloalkyl") or substituted by one or more substituents ("substituted cycloalkyl").
As used herein, the term "heterocyclyl" refers to a monovalent cyclic molecular structure (i.e., a radical of a heterocycle) that contains atoms of at least two different elements in one or more rings. See also: oxford Dictionary of Biochemistry and Molecular Biology, oxford University Press, oxford,1997, which is evidence that heterocycles are a well-recognized term in the field of organic chemistry.
The term "dipeptide" as used herein refers to a peptide consisting of two amino acid residues, which may be denoted herein as-a 1 -A 2 -. For example, the dipeptides used in the synthesis of the protease cleavable linker-payload conjugates disclosed herein may be selected from the group consisting of: val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Lys and Val-Ala.
As used herein, the term "halo" or "halogen" refers to F, cl, br or I.
The aryl or heteroaryl aromatic ring may be substituted at one or more ring positions with such substituents as described above, for example, alkyl, alkenyl, alkynyl, halogen, hydroxy, alkoxy, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylic acid, alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl, alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl, alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthio carbonyl, phosphate, phosphonyl, phosphinyl, amino (which includes alkylamino, dialkylamino, arylamino, diarylamino and alkylaryl amino), amido (which includes alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino, imino, mercapto, alkylthio, arylthio, thiocarboxyl, sulfate, alkylsulfinyl, sulfonate, sulfamoyl, sulfinylamino, nitro, trifluoromethyl, cyano, nitro, heterocyclyl, alkylaryl or aromatic or heteroaromatic moieties.
As used herein, the term "hydroxy" refers to a group of formula-OH.
As used herein, the term "hydroxyl" refers to hydroxyl radicals (OH).
As used herein, the term "oxo" refers to oxygen double bonded to carbon or another element (i.e., =o).
As used herein, the phrase "optionally substituted" means unsubstituted or substituted. Generally, the term "substituted" means that at least one hydrogen present on a group (e.g., a carbon or nitrogen atom) is replaced with a permissible substituent (e.g., a substituent which results in a stable compound upon substitution). The term "substituted" may include substitution with all permissible substituents of organic compounds such as any substituents described herein which result in the formation of stable compounds. For purposes of this disclosure, a heteroatom (e.g., nitrogen) may have a hydrogen substituent and/or any suitable substituent as described herein that satisfies the valency of the heteroatom and results in the formation of a stabilizing moiety.
As used herein, a "targeting ligand" is a molecule that can bind to a nanoparticle and target the nanoparticle to a tumor or cancer cell, typically by binding to the tumor or cancer cell (e.g., by binding to a protein expressed on the surface of the tumor or cancer cell). The targeting ligand may be any suitable molecule, for example, a small organic molecule (e.g., folic acid or folic acid analog), an antigen-binding portion of an antibody (e.g., fab fragment, fab ' fragment, F (ab ') 2 fragment, scFv fragment, fv fragment, dsFv diabody (diabody), dAb fragment, fd ' fragment, fd fragment, or an isolated Complementarity Determining Region (CDR) region), an antibody mimetic (e.g., aptamer, affibody, affinity protein (affilin), affinity peptide (affimer), anti-cardiolipin (anti-panatin), avimer, darpin, etc.), a binding domain of a receptor, a nucleic acid, a lipid, etc.
Furthermore, those skilled in the art will appreciate that the synthetic methods as described herein utilize a variety of protecting groups. As used herein, the term "protecting group" refers to a specific functional moiety, e.g., O, S or N, that is temporarily blocked so that the reaction can proceed selectively at another reaction site in the polyfunctional compound. The protecting groups are introduced or removed at appropriate stages in the synthesis of the compounds using methods known to those skilled in the art. Protecting groups are applied according to standard methods of organic synthesis described in the literature (Theodora W.Greene and Peter G.M.Wuts (2007) Protecting Groups in Organic Synthesis, 4 th edition, john Wiley and Sons, wherein the parts relating to protecting groups are incorporated herein by reference).
Exemplary protecting groups include, but are not limited to, oxygen, sulfur, nitrogen, and carbon protecting groups. For example, oxygen protecting groups include, but are not limited to, methyl ether, substituted methyl ether (e.g., MOM (methoxymethyl ether), MTM (methylthiomethyl ether), BOM (benzyloxymethyl ether), PMBM (heptoxybenzyloxymethyl ether)), optionally substituted ethyl ether, optionally substituted benzyl ether, silyl ether (e.g., TMS (trimethylsilyl ether), TES (triethylsilyl ether), TIPS (triisopropylsilyl ether), TBDMS (tert-butyldimethylsilyl ether), tribenzylsilyl ether, TBDPS (tert-butyldiphenylsilyl ether), esters (e.g., formate, acetate, benzoate (Bz), trifluoroacetate, dichloroacetate), carbonates, cyclic acetals, and ketals. Furthermore, nitrogen protecting groups include, but are not limited to, carbamates (including methyl, ethyl, and substituted carbamates (e.g., troc), amides, cyclic imide derivatives, N-alkyl and N-aryl amines, imine derivatives, and enamine derivatives, and the like. Amino protecting groups include, but are not limited to, fluorenylmethoxycarbonyl (Booc), carboxyl (fz), trifluoroacetamide groups, and the like.
Throughout this disclosure, nanoparticle Drug Conjugates (NDCs) may sometimes be referred to as CDCs (C' DOT-drug-conjugates), e.g., FA-CDCs, or simply functionalized nanoparticles.
Nanoparticles
The methods disclosed herein can be used to functionalize any suitable nanoparticle. For example, silica nanoparticles (which include nanoparticles composed partially of silica, or composed entirely of silica) may be functionalized using the methods disclosed herein. The nanoparticle may have a diameter of about 0.5nm to about 100nm, for example, about 0.1nm to about 50nm, about 0.5nm to about 25nm, about 1nm to about 20nm, about 0.8nm to about 15nm, about 1nm to about 10nm, or about 1nm to about 8nm, for example, about 1nm, about 2nm, about 3nm, about 4nm, about 5nm, about 6nm, about 7nm, about 8nm, about 9nm, about 10nm, about 11nm, or about 12 nm.
The nanoparticle may comprise a core surrounded by a shell (i.e., a core-shell nanoparticle). Alternatively, the nanoparticle may have only a core and no shell. The nanoparticle may also include a material that is reactive (e.g., on the surface of) silane moieties (e.g., silica surface). The shell of the nanomaterial may be a material that reacts with the silane moiety (e.g., a silica shell). The methods disclosed herein can be used to modify silica nanoparticles comprising a silica-based core and a silica-based shell surrounding at least a portion of the core (core-shell silica nanoparticles). The nanoparticle may be a non-mesoporous nanoparticle (e.g., a non-mesoporous core-shell nanoparticle), for example, a non-mesoporous silica nanoparticle (e.g., a non-mesoporous core-shell silica nanoparticle).
The nanoparticle may comprise an organic polymer coating on its surface. Organic polymers that may be attached to the nanoparticle include, but are not limited to, polyethylene glycol (PEG), polylactic acid esters (polylactates), polylactic acids (polylactic acids), sugars, lipids, polyglutamic acid (PGA), polyglycolic acid, poly (lactic-co-glycolic acid) (PLGA), polyvinyl acetate (PVA), and combinations thereof. For example, the nanoparticle may have a layer of polyethylene glycol (PEG) molecules attached to the surface. Certain organic polymer coatings can provide advantageous properties to nanoparticles, for example, properties suitable for biological systems. For example, after administration of a nanoparticle comprising a PEG coating to an animal (e.g., a human), the PEG groups can prevent serum proteins from adsorbing onto the nanoparticle, and can promote efficient urine excretion and reduce aggregation of the nanoparticle (see, e.g., burns et al, "Fluorescent silica nanoparticles with efficient urinary excretion for nanomedicine", nano Letters (2009) 9 (1): 442-448, which is incorporated herein by reference).
The shell of the nanoparticle may have a range of layers. For example, the shell may include from about 1 to about 20 layers, from about 1 to about 15 layers, from about 1 to about 10 layers, or from about 1 to about 5 layers. For example, the shell may include about 1 to about 3 layers. The thickness of the shell may range from about 0.5nm to about 90nm, for example, from about 1nm to about 40nm, from about 1nm to about 20nm, from about 1nm to about 10nm, or from about 1nm to about 5nm. For example, the thickness of the shell may be about 1nm to about 2nm. The shell of the nanoparticle may cover only a portion of the nanoparticle or the entire particle. For example, the shell may cover from about 1% to about 100%, from about 10% to about 80%, from about 20% to about 60%, or from about 30% to about 50% of the nanoparticle. The shell of the nanoparticle may comprise silica, and/or may be the reaction product of a silica forming compound, for example, a tetraalkyl orthosilicate, for example, tetraethyl orthosilicate (TEOS).
The nanoparticle (e.g., nanoparticle core and/or shell) may be substantially non-porous, mesoporous, semi-porous, or porous. The nanoparticle may comprise a non-porous surface and a porous surface. The bore surface may be referred to as an inner surface. The nanoparticle may also have a non-porous surface (or a non-porous surface). The non-porous surface may be referred to herein as the nanoparticle outer surface. The pore surface (e.g., at least a portion of the pore surface) and/or the non-porous surface (e.g., at least a portion of the non-porous surface) of the nanoparticle may be functionalized. As described above, the nanoparticle and/or nanoparticle shell may be non-mesoporous.
The nanoparticle may comprise a dye, for example, a fluorescent compound. The dye may be contained within the nanoparticle core. For example, the nanoparticle may comprise a dye covalently encapsulated in the nanoparticle. The dye in the silica nanoparticle may be the reaction product of a reactive dye (e.g., a fluorescent compound) and a co-reactive organosilane compound. The silica nanoparticles may comprise the reaction product of a reactive dye compound and a co-reactive organosilane compound and silica.
The nanoparticle may incorporate any known fluorescent compound, for example, a fluorescent organic compound, dye, pigment, or combination thereof. Such fluorescent compounds may be incorporated into the silica matrix of the silica nanoparticle core. Various suitable chemically reactive fluorescent dyes/fluorophores are known, see for example Molecular Probes Handbook of Fluorescent Probes and Research Chemicals, 6 th edition, r.p. haugland edit (1996). The fluorescent compound may be covalently encapsulated within the core of the nanoparticle.
The fluorescent compound may be, but is not limited to, a near infrared fluorescent (NIRF) dye located within the nanoparticle core that may provide higher brightness and fluorescence quantum yield relative to the free fluorescent dye. Near infrared emission probes are known to exhibit reduced tissue attenuation and autofluorescence (Burns et al, supra). Fluorescent compounds that may be used (e.g., encapsulated by NDC) in the present disclosure include, but are not limited to, cy5, cy5.5 (also known as Cy 5++), cy2, fluorescein Isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), phycoerythrin, cy7, fluorescein (FAM), cy3, cy3.5 (also known as Cy 3++), texas Red (sulfa 101 acyl chloride),-Red 640、/>red 705, tetramethyl rhodamine (TMR), rhodamine derivatives (ROX), hexachlorofluorescein (HEX), rhodamine 6G (R6G), rhodamineMing derivatives JA133, alexa fluorochromes (e.g. ALEXA +.>488、ALEXA/>546、ALEXA633、ALEXA />555 and ALEXA->647 4', 6-diamidino-2-phenylindole (DAPI), propidium iodide, aminomethylcoumarin (AMCA), spectrum Green, spectrum Orange, spectrum Aqua LISSAMINE) TM And fluorescent transition metal complexes such as europium. Fluorescent compounds that may be used also include fluorescent proteins, for example GFP (green fluorescent protein), enhanced GFP (EGFP), blue fluorescent proteins and their derivatives (BFP, EBFP, EBFP, chalcopyrite, mKalama 1), cyan fluorescent proteins and their derivatives (CFP, ECFP, cerulean, cyPet) and yellow fluorescent proteins and their derivatives (YFP, citrine, venus, YPet) (WO 2008/142571, WO2009/056282, WO 1999/22026). In a preferred aspect, the fluorescent compound is Cy5.
Nanoparticles can be synthesized by the following steps: (1) Covalently conjugating a fluorescent compound (e.g., a reactive fluorescent dye (e.g., cy 5)) with a reactive moiety (including, but not limited to, maleimide, iodoacetamide, thiosulfate, amine, N-hydroxysuccinimide ester, 4-sulfo-2, 3,5, 6-tetrafluorophenyl (STP) ester, sulfosuccinimidyl ester, sulfodichlorophenol ester, sulfonyl chloride, hydroxyl, isothiocyanate, carboxyl) to an organosilane compound (e.g., a co-reactive organosilane compound) to form a fluorescent silica precursor, and reacting the fluorescent silica precursor to form a fluorescent core; or (2) reacting the fluorescent silica precursor with a silica forming compound, such as a tetraalkoxysilane, to form a fluorescent core, and reacting the resulting fluorescent core with a silica forming compound, such as a tetraalkoxysilane, to form a silica shell on the core, thereby providing the fluorescent nanoparticle.
Fluorescent silica-based nanoparticles are known in the art and are described in U.S. patent No. 8,298,677;9,625,456;10,548,997;9,999,694;10,039,847 and 10,548,998; the contents of which are incorporated herein by reference in their entirety.
Nanoparticles functionalized by the methods disclosed herein can comprise nanoparticles comprising a silica-based core and a silica shell surrounding at least a portion of the core, and polyethylene glycol (PEG) covalently bonded to the nanoparticle surface. The nanoparticle may have a fluorescent compound covalently encapsulated within the nanoparticle core. For example, ultra-small pegylated silica nanoparticles (referred to as C' Dot) may be functionalized using the methods disclosed herein. C' Dot (see, e.g., ma, K. Et al, chemistry of Materials (2015) 27 (11): 4119-4133; the contents of which are incorporated herein by reference in their entirety) may be prepared as described previously. Nanoparticles for use in the methods described herein may also be as described in Ma, K, etc., chem. Ma, K, etc., chem. Mate (2016) 28:1537-1545; ma, K, et al chem.mate (2017) 29:6840-6855; the preparation is described in WO 2016/179260 and WO 2018/213851. Each C' Dot comprises pegylated silica particles (about 6nm in diameter) in which a near infrared fluorescent Cy5 dye is covalently encapsulated.
Method for functionalizing nanoparticles
Using the methods disclosed herein, the nanoparticles can be modified to incorporate one or more functional groups on the surface of the nanoparticle, which can be further modified (e.g., by conjugation of the functional groups to the molecule). For example, a material on the surface of a nanoparticle that is reacted with a silane (e.g., silanol groups) can be reacted with a bifunctional precursor described herein (e.g., a bifunctional precursor comprising a silane group and another functional group) to attach the other functional group to the nanoparticle surface. The material that reacts with the silane groups may be under the organic polymer layer coating of the nanoparticle (e.g., under the PEG layer), and the bifunctional precursor may be inserted into the interstitial spaces of the organic polymer molecules and react with the material on the surface of the nanoparticle.
Using the methods disclosed herein, the nanoparticles can be functionalized with one or more alkyne moieties, which can be further conjugated with compounds comprising alkyne reactive groups (e.g., azides, dienes, nitrones, or nitrile oxides). Thus, the methods disclosed herein may allow functionalization of nanoparticles for specific uses (e.g., for targeted cancer treatment). For example, a compound comprising an alkyne reactive group can also comprise a moiety that can target (e.g., have affinity for) a particular biological target (e.g., a cancer cell, e.g., a receptor expressed by a cancer cell). For example, the molecule may be a targeting ligand, e.g., a compound that binds a particular receptor, such as a compound that binds a folate receptor, e.g., folic acid or a derivative thereof. Thus, the methods disclosed herein can be used to functionalize nanoparticles suitable for targeting cancer cells. The compound comprising an alkyne reactive group can be a cytotoxic compound, and can comprise a cleavable linker, such that the cytotoxic compound can be released into a biological system (e.g., within a cancer cell) after the linker is cleaved. The payload may be a cytotoxic drug, for example, a small molecule chemotherapeutic drug, such as irinotecan or a derivative thereof, and the linker may be a protease cleavable linker. For example, the preparation of an exemplary Nanoparticle Drug Conjugate (NDC) comprising an ixabepilone payload and a folate targeting ligand using the methods disclosed herein is depicted in fig. 1.
The methods disclosed herein can be used to functionalize nanoparticles coated with organic polymers. For example, the methods described herein can be performed after the nanoparticles have been coated with polyethylene glycol (after pegylation). This reduces the need for reaction optimisation and minimises the changes required to the synthesis scheme and the nanoparticles themselves (e.g. no changes are required to optimise the surface chemistry of the nanoparticles prior to performing the methods disclosed herein), which is more efficient than conventional methods of nanoparticle functionalization. The introduction of functional groups into nanoparticles having organic polymer coatings (e.g., PEG) is often not possible or very inefficient using conventional methods of nanoparticle functionalization, in part because of the steric hindrance of the organic polymer, which prevents conjugation of the precursor molecule to the nanoparticle surface, which often provide low yields or fail to provide any functionalized nanoparticles. A typical approach to avoid these problems for functionalizing nanoparticles is to introduce precursor molecules (containing the desired functional groups) to the nanoparticle surface while adding organic polymer coating molecules. However, this approach has significant drawbacks such as unpredictable reaction kinetics (e.g., due to attraction or repulsion between the functional groups of the precursor molecules and the nanoparticle surface), the "embedding" of the functional groups into the organic polymer layer of the nanoparticle during synthesis, and aggregation of the partially coated nanoparticle during synthesis, each of which can lead to low yields and suboptimal nanoparticle functionalization. This is particularly problematic when silica nanoparticles having charged surfaces are used, which exacerbate unwanted intermolecular interactions and affect reaction kinetics in an unpredictable manner. The methods disclosed herein can overcome these problems.
Furthermore, the methods disclosed herein provide a solution to the problem of how to attach macromolecules (e.g., DBCO) to the surface of sterically hindered nanoparticles that are coated with an organic polymer (e.g., a PEG layer). Certain molecules, particularly those with larger size and steric hindrance, have limited ability to enter the nanoparticle surface, resulting in lower reactivity and impeding attachment of the molecule to the nanoparticle surface. For example, when using DBCO-PEG-silane as a bifunctional precursor, only two DBCO groups may be attached to the nanoparticle even when the proportion of DBCO-PEG-silane precursor in the reaction mixture is relatively high (see, e.g., example 1 and fig. 2).
The disclosed methods may involve a two-step reaction sequence in which a small first bifunctional precursor (e.g., a precursor comprising silane groups) having a relatively high diffusivity is first bound to the nanoparticle surface, followed by subsequent modification with another compound (e.g., an additional bifunctional precursor). For example, a small bifunctional precursor comprising a silane moiety and another reactive group (e.g., diene, amine, thiol, hydroxyl, azide, alkene, nitrone, nitrile oxide, or alkyne) may be added to a solution of nanoparticles comprising a surface reactive with the silane group under conditions suitable for a reaction between the nanoparticle surface and the silane group, thereby providing nanoparticles functionalized with the other reactive group.
Conditions suitable for the reaction between the silane moiety and the nanoparticle surface may include combining the nanoparticle and the first bifunctional precursor in an aqueous reaction medium (e.g., a reaction medium that is substantially water). The reaction medium may also comprise an aprotic organic solvent, such as dimethyl sulfoxide (DMSO) or acetonitrile. For example, the reaction medium may contain no more than 20v/v% aprotic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% aprotic solvent. The reaction medium may also comprise a protic organic solvent, for example an alcohol (such as t-butanol). For example, the reaction medium may contain no more than 20v/v% of a protic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% of a protic solvent. The reaction medium may comprise a buffer (e.g., an aqueous buffer), for example, phosphate Buffered Saline (PBS). For example, the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration of between about 1x and about 5x (e.g., about 1x, about 2x, about 3x, about 4x, or about 5 x). The reaction medium may be substantially free of organic solvents (e.g., substantially free of organic protic solvents, substantially free of aprotic solvents, or substantially free of any organic solvents). The reaction medium may be substantially free of buffer. The reaction medium may be substantially water.
Conditions suitable for the reaction between the silane moiety and the nanoparticle surface may include combining the nanoparticle and the first bifunctional precursor in the reaction medium for a time period, e.g., from about 1 minute to about 60 minutes, from about 0.5 hour to about 24 hours, from about 0.5 hour to about 18 hours, from about 0.5 hour to about 12 hours, from about 0.5 hour to about 6 hours, from about 0.5 hour to about 4 hours, from about 0.5 hour to about 3 hours, from about 0.5 hour to about 2 hours, from about 0.5 hour to about 1 hour, or from about 1 hour to about 48 hours. For example, the conditions may include maintaining the reaction medium overnight.
Conditions suitable for the reaction between the silane moiety and the nanoparticle surface may include combining the nanoparticle and the first bifunctional precursor in a reaction medium and heating the reaction medium to, for example, a temperature of about 20 ℃ or greater, e.g., about 35 ℃ or greater, about 40 ℃ or greater, about 45 ℃ or greater, about 50 ℃ or greater, about 60 ℃ or greater, about 70 ℃ or greater, about 80 ℃ or greater, about 90 ℃ or greater, about 100 ℃ or greater, about 110 ℃ or greater, or about 120 ℃ or greater, e.g., about 20 ℃ to about 60 ℃, about 40 ℃ to about 80 ℃, about 60 ℃ and about 100 ℃, about 20 ℃ and about 40 ℃, about 30 ℃ and about 50 ℃, about 40 ℃ and about 60 ℃, about 50 ℃ to about 70 ℃, about 60 ℃ to about 80 ℃, about 70 ℃ to about 90 ℃, about 80 ℃ to about 100 ℃, or about 90 ℃ to about 110 ℃. The conditions may include maintaining the nanoparticle and the first bifunctional precursor at room temperature. Conditions suitable for the reaction between the silane moiety and the nanoparticle surface may include stirring, shaking or application of other mixing methods to the reaction medium.
The first bifunctional precursor may be any suitable silane-containing compound that reacts with the nanoparticle surface and may comprise functional groups that may be used for further modification (e.g., after the first bifunctional precursor is attached to the nanoparticle). The functional groups may be groups that are non-reactive with the nanoparticle and/or non-reactive with the silane. The first bifunctional precursor may comprise an alkylene (e.g., C 1 -C 6 Alkylene) having a silane group at one end and another functional group at the other end (e.g., diene, amine, thiol, hydroxyl, azide, alkene, nitrone, nitrile oxide, or alkyne). The first bifunctional precursor may comprise a heteroalkylene (e.g., C 1 -C 6 Heteroalkylene) having a silane group at one end and another functional group at the other end (e.g., diene, amine, thiol, hydroxyl, azide, alkene, nitrone, nitrile oxide, or alkyne). First pair ofThe functional precursor may comprise an alkylene group (e.g., C 1 -C 6 Alkylene) having a silane group at one end and a diene group at the other end, for example, a cyclodienyl group (e.g., a cyclopentadienyl group). Any suitable diene group may be used. For example, any diene suitable for Diels-Alder (or heter-Diels Alder) cycloaddition may be used. Examples of dienes that may be present in the first difunctional precursor include, but are not limited to, cyclopentadiene, cyclohexadiene, furan, butadiene, and derivatives thereof. In addition, for example, other moieties that can be modified by reaction with another entity may be present on the first bifunctional precursor, and the method is not limited to the use of Diels-Alder cycloadditions (e.g., other cycloadditions, or other bonding reactions, such as those described herein, may be used).
The first bifunctional precursor may comprise a structure of formula (a):
wherein R is 1 Is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen, -OR A 、-NR B R C 、-NO 2 or-CN, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group may be substituted or unsubstituted; each R 2 Independently hydrogen, alkyl, halogen OR-OR A ;R A 、R B And R is C Each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; n is an integer from 0 to 12; m is an integer from 0 to 5. For example, R 1 Can be alkyl, C3-alkenyl, C3-alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen, -OR A Wherein each alkyl, C3-alkenyl, C3-alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl group may be substituted or unsubstituted; each R 2 Independently hydrogen, alkyl, halogen OR-OR A ;R A Is hydrogen or alkylA group, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; n is an integer from 0 to 12; m is an integer from 0 to 5. For example, R 2 Can be-OR A Wherein R is A Alkyl (e.g., methyl or ethyl); n may be an integer from 1 to 5 (e.g., 3); m may be 0. In some aspects, R 2 is-OEt; n is 3 and m is 0. For example, the first bifunctional precursor may comprise a structure of formula (A-1):
the first difunctional precursor may be (3-cyclopentadienyl propyl) triethoxysilane (e.g., as shown in example 3), wherein the method provides nanoparticles functionalized with one or more cyclopentadienyl groups. Alternatively, the first bifunctional precursor may be (3-aminopropyl) triethoxysilane (e.g., as shown in example 2), wherein the method provides nanoparticles functionalized with one or more amine groups.
The step of reacting the nanoparticle with the first bifunctional precursor may provide a nanoparticle functionalized with a first reactive group, which may be further modified. For example, a nanoparticle functionalized with a first reactive group may be contacted with a second bifunctional precursor comprising a moiety reactive with the first reactive group.
The contacting of the nanoparticle functionalized with the first reactive group with the second bifunctional precursor may be performed under any conditions suitable for the reaction between the first reactive group and the moiety reactive with the first reactive group. For example, conditions suitable for the reaction between the first reactive group and the moiety that reacts with the first reactive group may include an aqueous reaction medium (e.g., a reaction medium that is substantially water). The reaction medium may comprise a buffer (e.g., an aqueous buffer), for example, phosphate Buffered Saline (PBS). For example, the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration of between about 1x and about 5x (e.g., about 1x, about 2x, about 3x, about 4x, or about 5 x). The reaction medium may also comprise an aprotic organic solvent, such as dimethyl sulfoxide (DMSO) or acetonitrile. For example, the reaction medium may contain no more than 20v/v% aprotic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% aprotic solvent. The reaction medium may also comprise a protic organic solvent, for example an alcohol (such as t-butanol). For example, the reaction medium may contain no more than 20v/v% of a protic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% of a protic solvent. The reaction medium may be substantially free of organic solvents (e.g., substantially free of organic protic solvents, substantially free of aprotic solvents, or substantially free of any organic solvents). The reaction medium may be substantially water.
Conditions suitable for the reaction between the first reactive group and the moiety reacted with the first reactive group may include maintaining the reaction mixture for a time, for example, from about 1 minute to about 60 minutes, from about 0.5 hour to about 24 hours, from about 0.5 hour to about 18 hours, from about 0.5 hour to about 12 hours, from about 0.5 hour to about 6 hours, from about 0.5 hour to about 4 hours, from about 0.5 hour to about 3 hours, from about 0.5 hour to about 2 hours, from about 0.5 hour to about 1 hour, or from about 1 hour to about 48 hours. For example, the conditions may include maintaining the reaction medium overnight.
Conditions suitable for the reaction between the first reactive group and the moiety reacted with the first reactive group may include heating the reaction medium to, for example, a temperature of about 20 ℃ or greater, for example, about 35 ℃ or greater, about 40 ℃ or greater, about 45 ℃ or greater, about 50 ℃ or greater, about 60 ℃ or greater, about 70 ℃ or greater, about 80 ℃ or greater, about 90 ℃ or greater, about 100 ℃ or greater, about 110 ℃ or greater, or about 120 ℃ or greater, for example, about 20 ℃ to about 60 ℃, about 40 ℃ to about 80 ℃, about 60 ℃ and about 100 ℃, about 20 ℃ to about 40 ℃, about 30 ℃ and about 50 ℃, about 40 ℃ and about 60 ℃, about 50 ℃ to about 70 ℃, about 60 ℃ to about 80 ℃, about 70 ℃ to about 90 ℃, about 80 ℃ to about 100 ℃, or about 90 ℃ to about 110 ℃. The conditions may include maintaining the reaction mixture at room temperature. Conditions suitable for the reaction between the first reactive group and the moiety reactive with the first reactive group may also include stirring, shaking or application of other mixing methods to the reaction medium.
The first reactive group can be a diene (e.g., cyclopentadiene), and the second bifunctional precursor can comprise a dienophile (e.g., a dienophile as described herein, e.g., maleimide) that reacts with the first reactive group, wherein the contacting can be conducted under conditions sufficient to promote a Diels-Alder cycloaddition reaction between the diene and the dienophile.
The dienophile may be any suitable dienophile, for example, a moiety comprising an electron deficient olefin. The dienophile may include a cyclic dienophile, such as maleimide, quinone, maleic anhydride, dialkylacetylene dicarboxylic acid, or any derivative thereof.
It is to be understood that the positions of the diene and dienophile may be on the first dual function precursor or the second dual function precursor, respectively. In other words, while certain methods exemplified herein are characterized by a first bifunctional precursor comprising a diene and a second bifunctional precursor comprising a dienophile, these methods or precursors can be readily modified such that the first bifunctional precursor comprises a dienophile and the second bifunctional precursor comprises a diene. Furthermore, while the reaction between diene and dienophile is exemplified in this disclosure, conjugation between the first reactive group and the second bifunctional precursor may involve any other suitable covalent bond forming reaction, which may be determined by selection of groups on the first bifunctional precursor and the second bifunctional precursor. For example, the methods of functionalizing nanoparticles disclosed herein (e.g., at any step of the method) may involve etherification, amide bond formation, click chemistry, diels-Alder cycloaddition, hetero-Diels-Alder cycloaddition, 1, 2-addition (e.g., michael addition), huisgen cycloaddition, nitrone-olefin cycloaddition, 3+2 cycloaddition, 4+2 cycloaddition, olefin metathesis, or midkolb et al, angelw.chem.int.ed. (2001) 40:2004-2021 (which is incorporated herein by reference in its entirety). For example, rather than using a Diels-Alder reaction to conjugate a second bifunctional precursor to a nanoparticle via a first reactive group, the method is modified such that the first reactive group (introduced by the first bifunctional precursor) may be an amine, and the second bifunctional precursor may comprise an N-hydroxysuccinimide (NHS) ester group, and the second bifunctional precursor and the functionalized nanoparticle comprising an amine group may be contacted under conditions sufficient to promote an amine-ester reaction, and a bond is formed between the first reactive group and the second bifunctional precursor. Alternatively, rather than using a Diels-Alder reaction to conjugate a second bifunctional precursor to the nanoparticle via a first reactive group, a click chemistry reaction may be used, for example, wherein the first reactive group (introduced by the first bifunctional precursor) is an azide or alkyne and the second bifunctional precursor is an alkyne or azide, and the second bifunctional precursor and the functionalized nanoparticle may be contacted under conditions sufficient to promote a click chemistry reaction between the first reactive group and the second bifunctional precursor.
In a particular aspect, the first bifunctional precursor comprises a diene (e.g., cyclopentadiene), and the second bifunctional precursor comprises a dienophile (e.g., maleimide) that can react with the diene in a Diels-Alder reaction to attach the second bifunctional precursor to the nanoparticle (via the first bifunctional precursor), as demonstrated by example 3, as also depicted in scheme 1.
The second dual-function precursor may comprise a further reactive group, for example, on the end of the second dual-function precursor opposite the portion that reacts with the first reactive group. For example, the second bifunctional precursor may comprise a group at one end that is reactive with a diene moiety (e.g., a dienophile such as maleimide) and another group at another end that is suitable for further modification. The other reactive group (sometimes referred to as a functional group) on the second dual-function precursor may be a moiety that reacts with an alkyne, azide, diene, nitrone, or nitrile oxide. The reactive group may be a moiety suitable for performing click chemistry reactions. For example, the reactive group may include an alkyne, azide, diene, nitrone, or nitrile oxide. The alkyne can be a strained alkyne (strained alkyne). For example, the reactive group may include a Dibenzoazacyclooctyne (DBCO) group or a derivative thereof (sometimes referred to as a DIBAC). The alkyne can include a Dibenzocyclooctynyl (DIBO) or derivative thereof. The alkyne can also be a terminal alkyne. Alternatively, the reactive group on the second bifunctional precursor may be any other group suitable for conjugation, for example, a diene, an amine, a thiol, a hydroxyl, an azide, a nitrone, a nitrile oxide, an alkene, or an alkene.
The second bifunctional precursor may also comprise a divalent linker, e.g., an alkylene or heteroalkylene, where the alkylene or heteroalkylene is unsubstituted or substituted (e.g., an alkylene or heteroalkylene substituted with one or more oxo groups). The divalent linker may comprise a PEG group, for example, PEG4. The divalent linker may comprise an amide bond, for example, one, two, three or more amide bonds. Each amide bond may be in any orientation. For example, an amide bond may be located in a divalent linker between a PEG group and an alkylene group, wherein the carbon atom of the carbonyl group of the amide is covalently attached to an atom of the PEG group, and the nitrogen atom of the amide is covalently attached to an atom of the alkylene group; or the carbon atom of the carbonyl group of the amide may be attached to an atom of the alkylene group and the nitrogen atom of the amide group may be attached to an atom of the PEG group. The divalent linker (e.g., heteroalkylene) can include a dienophile moiety at one terminus and an alkyne, e.g., a DBCO group, at the other terminus. Alternatively, the second bifunctional precursor may comprise a divalent linker (e.g., a heteroalkylene) that is at one terminus a NHS ester and at the other terminus an alkyne, e.g., a DBCO group. The divalent linker can comprise a PEG group (e.g., a PEG4 moiety), an amide group, or a combination thereof.
The second dual-function precursor may comprise a structure of formula (B)
Wherein: x is a reactive group, for example, a dienophile (e.g., a moiety comprising an electron deficient alkene group; or a cyclic dienophile, for example, maleimide, quinone, or maleic anhydride); y is a divalent linker (e.g., substituted or unsubstituted alkylene or heteroalkyleneAn alkyl group); r is R 3 And R is 4 Each independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen, -OR A 、-NR B R C 、-NO 2 or-CN, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl group may be substituted or unsubstituted; r is R A 、R B 、R C Each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; q and p are each independently integers from 0 to 4; and v is an integer from 0 to 2. For example, X may be maleimide; q and p may each independently be an integer 0; and v may be an integer of 1.
The divalent linker may comprise a structure of formula (G):
wherein A is 1 、A 2 And A 3 Each independently selected from the group consisting of: alkylene, alkenylene, alkynylene, heteroalkylene, cycloalkylene, arylene, heteroarylene, -C (O) -, -C (O) N (R) B )-、-N(R B )C(O)-、-N(R B ) -, -OC (O) -and-C (O) O-; r is R B Independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; k. v, w, x are each independently integers from 0 to 10; and each is provided withIndependently represents a point of attachment to another portion of the second dual function precursor. For example, A 1 And A 2 Can each independently be-C (O) N (R) B )-;A 3 Can be-C (O) O-; r is R B Independently hydrogen; k. v and w may each independently be an integer of 2; and x may be an integer of 4.
The divalent linker may comprise a structure of formula (C):
wherein each R is B Independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer from 0 to 10; and each is provided withIndependently represents a point of attachment to another portion of the second bifunctional precursor (e.g., to the dienophile or to a nitrogen atom of a heterocycle of DBCO).
The divalent linker may comprise a structure of formula (C):
wherein each R is B Independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer from 0 to 10; and each is provided withIndependently represents a point of attachment to another portion of the second bifunctional precursor (e.g., to the dienophile or to a nitrogen atom of a heterocycle of DBCO).
The divalent linker may comprise a structure of formula (C "):
wherein each R is B Independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl; x is an integer from 0 to 10; and each is provided withIndependently represents a point of attachment to another moiety of the second dual-function precursor (e.g., to a nitrogen atom of a NHS moiety, or to a nitrogen atom of a heterocycle of DBCO)。
The second dual-function precursor may comprise a structure of formula (B-1)
Where x is an integer from 0 to 10 (e.g., 4). For example, x may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a preferred embodiment, x is 4.
Reacting a diene moiety (e.g., a diene moiety covalently attached to the nanoparticle surface) with a second bifunctional precursor can provide a compound of formula (NP-1)
Where x is an integer from 0 to 10 (e.g., 4), and where the silicon atom is part of the nanoparticle. For example, x may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. In a preferred embodiment, x is 4.
The efficiency of the reaction between the second dual-function precursor (e.g., a DBCO-containing precursor such as DBCO-maleimide) and the functionalized nanoparticle (e.g., a nanoparticle comprising a diene moiety) may be independent of the diffusion coefficient of the second dual-function precursor. For example, the second bifunctional precursors disclosed herein comprising PEG groups of different lengths may have similar reactivity to functionalized nanoparticles regardless of the length of the PEG (see, e.g., example 4 and fig. 2, wherein the use of DBCO-PEG-maleimide precursors with PEG of different lengths does not significantly affect the reactivity). This is in contrast to typical processes involving other precursors (e.g., as shown in schemes 3 or 4), where the reaction efficiency can be greatly affected by the size or molecular mass of the precursor. The bifunctional precursors disclosed herein (e.g., the second bifunctional precursor) may also have a particular solubility or polarity that may avoid undesired electrostatic interactions (e.g., repulsion) with silanol groups (e.g., silanol groups on another precursor or nanoparticle). Furthermore, the bifunctional precursors disclosed herein (e.g., the second bifunctional precursor) may have a specific length (e.g., calculated as a PEG spacer) to impart desired properties to the resulting functionalized nanoparticle (after conjugation to the second bifunctional precursor), e.g., improved accessibility for subsequent modification by another precursor, e.g., using click chemistry.
The methods disclosed herein can be used to provide nanoparticles suitable for particular applications, such as therapeutic applications. For example, the method can be used to provide nanoparticles suitable for targeting biological targets, e.g., for therapy (e.g., cancer therapy), surgical navigation, imaging, diagnosis, or a combination thereof. The nanoparticle may be suitable for in vivo use, or may be used ex vivo, for example, for analytical or diagnostic applications. The methods disclosed herein can be used to prepare nanoparticles suitable for targeting a particular receptor (or group of receptors) in a biological system. The method can also be used to prepare functionalized nanoparticles (e.g., NDCs) suitable for delivering a payload to a biological target (or targets). For example, using the methods disclosed herein, the nanoparticle can be conjugated to a targeting ligand and conjugated to a payload moiety. For example, the methods disclosed herein can be used to prepare folate receptor targeted NDCs, such as for targeted cancer treatments, e.g., the exemplary NDCs depicted in fig. 1.
Reacting the nanoparticle with a second bifunctional precursor may provide a nanoparticle comprising one or more groups suitable for conjugation to a molecule (e.g., a targeting ligand and/or payload). For example, reacting the nanoparticle with a second bifunctional precursor comprising an alkyne group may provide a nanoparticle functionalized with one or more alkyne groups, wherein the alkyne groups may be further conjugated with a compound comprising alkyne reactive groups.
Any desired compound comprising a reactive group may be attached to the nanoparticles disclosed herein functionalized with the appropriate corresponding reactive group. For example, any desired compound comprising an alkyne reactive group can be attached to the nanoparticle functionalized with an alkyne. For example, a payload moiety (e.g., comprising a cytotoxic drug, e.g., isatecan) or a linker-payload conjugate comprising an alkyne reactive group can be conjugated to the nanoparticle. Alternatively, a targeting ligand comprising an alkyne reactive group (e.g., a Folate Receptor (FR) -targeting ligand, e.g., folic acid) can be conjugated to the nanoparticle. Any combination of compounds can be conjugated to the nanoparticle using this method. For example, both the targeting ligand and the payload moiety may be conjugated to the nanoparticle. Other molecules, e.g., labels (e.g., radiolabels or dyes), polymers, and/or macromolecules, can also be conjugated to the nanoparticles using this method.
For example, nanoparticles comprising alkyne moieties (e.g., prepared using the methods disclosed herein) can be contacted with a compound comprising alkyne reactive groups (e.g., azide, diene, nitrone, or nitrile oxide) under conditions suitable for a reaction between alkyne moieties and alkyne reactive groups, thereby forming nanoparticles functionalized with a compound (e.g., a compound comprising a payload or targeting ligand).
Conditions suitable for the reaction between the alkyne moiety and the alkyne reactive group may include any suitable click chemistry conditions (see, e.g., kolb et al, angel. Chem. Int. Ed. (2001) 40:2004-2021). For example, promoting the reaction between the alkyne moiety and the alkyne reactive group can include reacting the nanoparticle comprising the alkyne moiety and the compound comprising the alkyne reactive group with a catalyst (e.g., a copper catalyst, such as CuSO 4 、CuI、CuCl、Cu(OAc) 2 、CuSO 2 CuBr). Other metal catalysts known to affect click chemistry, such as ruthenium catalysts, may also be used. Alternatively, the reaction may be carried out without a catalyst (i.e., without catalyst conditions, such as copper-free conditions). For example, alkynes on functionalized nanoparticles may be suitable for catalyst-free click chemistry reactions (e.g., strain-promoted cycloaddition). For example, DBCO may be used in a click chemistry reaction without a catalyst.
Conditions suitable for the reaction between the alkyne moiety and the alkyne reactive group can include combining the nanoparticle comprising the alkyne moiety and the compound comprising the alkyne reactive group in an aqueous reaction medium (e.g., a reaction medium that is substantially water). The reaction medium may comprise a buffer (e.g., an aqueous buffer), for example, phosphate Buffered Saline (PBS). For example, the reaction medium may comprise a buffer (e.g., an aqueous buffer, such as PBS) at a final concentration of between about 1x and about 5x (e.g., about 1x, about 2x, about 3x, about 4x, or about 5 x). The reaction medium may also comprise aprotic organic solvents, such as DMSO or acetonitrile. For example, the reaction medium may contain no more than 20v/v% aprotic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% aprotic solvent. The reaction medium may also comprise a protic organic solvent, for example an alcohol (such as t-butanol). For example, the reaction medium may contain no more than 20v/v% of a protic solvent, e.g., less than 18v/v%, less than 16v/v%, less than 14v/v%, less than 12v/v%, less than 10v/v%, less than 8v/v%, less than 6v/v%, less than 4v/v%, less than 2v/v%, or less than 1v/v% of a protic solvent. The reaction medium may be substantially free of organic solvents (e.g., substantially free of organic protic solvents, substantially free of aprotic solvents, or substantially free of any organic solvents). The reaction medium may be substantially water.
Conditions suitable for the reaction between the alkyne moiety and the alkyne reactive group can include combining the nanoparticle comprising the alkyne moiety and the compound comprising the alkyne reactive group in the reaction medium for a time, for example, from about 1 minute to about 60 minutes, from about 0.5 hours to about 24 hours, from about 0.5 hours to about 18 hours, from about 0.5 hours to about 12 hours, from about 0.5 hours to about 6 hours, from about 0.5 hours to about 4 hours, from about 0.5 hours to about 3 hours, from about 0.5 hours to about 2 hours, from about 0.5 hours to about 1 hour, or from about 1 hour to about 48 hours. For example, the reaction medium is kept overnight.
Conditions suitable for the reaction between the alkyne moiety and the alkyne reactive group can include combining the nanoparticle comprising the alkyne moiety and the compound comprising the alkyne reactive group in a reaction medium and heating the reaction medium to, for example, about 20 ℃ or higher, e.g., about 35 ℃ or higher, about 40 ℃ or higher, about 45 ℃ or higher, about 50 ℃ or higher, about 60 ℃ or higher, about 70 ℃ or higher, about 80 ℃ or higher, about 90 ℃ or higher, about 100 ℃ or higher, about 110 ℃ or higher, or about 120 ℃ or higher, e.g., about 20 ℃ to about 60 ℃, about 40 ℃ to about 80 ℃, about 60 ℃ and about 100 ℃, about 20 ℃ and about 40 ℃, about 30 ℃ and about 50 ℃, about 40 ℃ and about 60 ℃, about 50 ℃ to about 70 ℃, about 60 ℃ to about 80 ℃, about 70 ℃ to about 90 ℃, about 80 ℃ to about 100 ℃, or about 90 ℃ to about 110 ℃. Alternatively, the reaction conditions may include maintaining the reaction mixture at room temperature. Conditions suitable for the reaction between the first reactive group and the moiety reactive with the first reactive group may also include stirring, shaking or application of other mixing methods to the reaction medium.
The compound comprising a reactive group (e.g., an alkyne reactive group) can comprise a targeting ligand, and can further comprise a linker (e.g., a non-cleavable linker). The targeting ligand may be a folate receptor targeting ligand. For example, the targeting ligand may be a molecule that can bind to a folate receptor (e.g., a folate receptor expressed on a cancer cell or tumor). The Folate Receptor (FR) targeting ligand may be folic acid, dihydrofolic acid, tetrahydrofolic acid, or folate receptor binding derivatives thereof. The folate receptor targeting ligand may be a macromolecule, e.g., a protein, peptide, aptamer, antibody or antibody fragment that can target the folate receptor. For example, the folate receptor targeting ligand may comprise a portion of an intact antibody, e.g., such as an antigen binding or variable region of an antibody. Examples of folate receptor targeted antibody fragments include, but are not limited to, fab fragments, fab ' fragments, F (ab ') 2 fragments, scFv fragments, fv fragments, dsFv diabodies, dAb fragments, fd ' fragments, fd fragments, or isolated Complementarity Determining Region (CDR) regions. Antigen binding fragments of antibodies can be produced by any means. For example, an antigen binding fragment of an antibody may be produced enzymatically or chemically by fragmentation of the whole antibody and/or it may be produced by genetic recombination encoding part of the antibody sequence. Alternatively or additionally, antigen binding fragments of antibodies may be produced wholly or partially synthetically. The linker may be any suitable divalent linker, for example, alkylene or heteroalkylene. The divalent linker may be a PEG group (e.g., a PEG1, PEG2, PEG3, or PEG4 group).
The compound comprising a reactive group (e.g., an alkyne reactive group) can comprise a structure of formula (D'):(D'), wherein J is a reactive group, e.g., an alkyne reactive group; for example, azides, dienes, nitrones or nitrile oxides; t is a targeting ligand (e.g., a folate receptor targeting ligand, e.g., folic acid or a derivative thereof); y is an integer from 0 to 20 (e.g., 3). For example, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
The alkyne reactive group containing compound can comprise the structure of formula (D):(D) Wherein T is a targeting ligand (e.g., a folate receptor targeting ligand, e.g., folic acid or a derivative thereof); and y is an integer from 0 to 20 (e.g., 3). For example, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
The compound comprising a reactive group (e.g., an alkyne reactive group) can comprise a structure of formula (D-1'):
wherein J is a reactive group (e.g., an alkyne reactive group; e.g., azide, diene, nitrone, or nitrile oxide); y is an integer from 0 to 20 (e.g., 3). For example, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
The alkyne reactive group containing compound may comprise the structure of formula (D-1):
where y is an integer from 0 to 20 (e.g., 3). For example, y may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 2, 3, or 4.
Compounds comprising a reactive group (e.g., an alkyne reactive group) can comprise a payload portion, and can further comprise a linker (e.g., a cleavable linker). The payload moiety may be a molecule having therapeutic utility, for example, for cancer treatment. For example, the payload moiety may be a cytotoxic compound, e.g., a small molecule having cytotoxic properties.
Exemplary payloads include enzyme inhibitors (see, e.g., "A Review of Evaluation of Enzyme Inhibitors in Drug Discovery," Robert a. Copeland, john Wiley and Sons, hoboken, new Jersey), e.g., topoisomerase inhibitors (e.g., i Sha Tikang, SN-38, topotecan, irinotecan, camptothecins, belotecan, indenoisoquinoline, phenanthridine, indolocarbazole, and the like), dihydrofolate reductase inhibitors, thymidylate synthase inhibitors, DNA intercalators, DNA minor groove binders, tubulin disrupters, DNA cleaving agents, anthracyclines, vinca drugs, mitomycins (e.g., mitomycin-C, mitomycin-a), bleomycin, cytotoxic nucleosides, pteridines, diyne, enediyne, podophyllotoxins, dolastatins (dolastastatins), auristatins (e.g., monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF)), maytansine, differentiation inducers, multicarubicins, and taxanes (e.g., paclitaxel). For example, the payload may be i Sha Tikang, SN-38, topotecan, irinotecan, belotecan, 9-aminocamptothecin, etoposide, camptothecin, paclitaxel, epothilone (esperamicin), 1, 8-dihydroxy-bicyclo [7.3.1] tridec-4-9-diene-2, 6-diyn-13-one, podophyllotoxin, aminoguanidine (anguidine), vincristine, vinblastine, duocarmycin, pyrrolobenzodiazepine, morpholino-doxorubicin, N- (5, 5-diacetoxy-pentyl) doxorubicin (a compound described in U.S. Pat. No. 5,198,560), daunorubicin, doxorubicin, aminopterin, actinomycin, bleomycin, N8-acetylspermidine, pyrrolobenzodiazepine, l- (2-chloroethyl) -l, 2-dimethyl sulfonyl, tacalcin (talsonin), doxorubicin, oxazidine, aureomycin, thiotepa, or derivatives thereof.
It will be appreciated that for the purpose of preparing the conjugates of the present disclosure, chemical modifications may be made to the desired payload in order to allow the payload to react more conveniently with the linker. For example, a functional group (e.g., an amine, hydroxyl, or sulfhydryl) may be pendant from a drug at a position that has minimal or acceptable impact on the activity or other properties of the drug.
The linker may be any suitable divalent linker and may comprise a moiety that may be cleaved under certain conditions. For example, the linker may be a self-destructing linker capable of releasing the payload in vitro or in vivo under certain conditions. The linker may be a protease cleavable linker (e.g., cleavable by a protease such as cathepsin or trypsin), a pH-sensitive linker, or a redox-sensitive linker. The protease cleavable linker may be a cathepsin B (Cat-B) cleavable linker. The linker can comprise a peptide moiety (e.g., a dipeptide), a PEG spacer, and/or a self-destructing moiety (e.g., p-aminobenzyloxycarbonyl (PABC)), e.g., for efficient cleavage in a cell (e.g., a cancer cell). The cleavable linker can be selectively cleaved at a desired location or after a selected time (e.g., after entry into a targeted cancer cell).
The compound comprising a reactive group (e.g., an alkyne reactive group) can comprise a structure of formula (E'):(E'), wherein J is a reactive group (e.g., an alkyne reactive group; e.g., azide, diene, nitrone, or nitrile oxide); l is a cleavable linker moiety (e.g., a protease cleavable linker moiety); p is a payload moiety (e.g., a cytotoxic drug, e.g., irinotecan); and y is an integer from 0 to 20 (e.g., 9). For example, L may be a protease cleavable linkerSeparately, P may be irinotecan, and y may be an integer from 0 to 20 (e.g., 7, 8, 9, 10, or 11).
The alkyne reactive group containing compound can comprise the structure of formula (E):(E) Wherein L is a cleavable linker moiety (e.g., a protease cleavable linker moiety); p is a payload moiety (e.g., a cytotoxic drug, e.g., irinotecan); y is an integer from 0 to 20 (e.g., 9). For example, L may be a protease cleavable linker moiety, P may be irinotecan, and y may be an integer from 0 to 20 (e.g., 7, 8, 9, 10, or 11).
The cleavable linker portion may comprise a structure of formula (F):
[AA]is a natural or unnatural amino acid residue; z is an integer from 1 to 5; w is an integer from 1 to 4 (e.g., 2 or 3); and each is provided with Independently represents a point of attachment to another portion of the compound comprising a reactive group (e.g., an alkyne reactive group; e.g., attachment to a PEG group, attachment to an alkyne reactive group, or attachment to a payload portion). For example, - [ AA] w May include Val-Lys, val-Cit, phe-Lys, trp-Lys, asp-Lys, val-Arg or Val-Ala, and z may be 2, one of which +.>Represents a point of attachment to the oxygen atom of the PEG group and the other +.>Represents the point of attachment to the nitrogen atom of irinotecan.
The cleavable linker portion may comprise a structure of formula (F-1):
one of which is a plateRepresents a point of attachment to another portion of the compound comprising a reactive group (e.g., an alkyne reactive group; e.g., attachment to a PEG group, attachment to an alkyne reactive group, or attachment to a payload portion). For example, a +.>Represents a point of attachment to the oxygen atom of the PEG group and the other +.>Represents the point of attachment to the nitrogen atom of irinotecan.
The compound comprising a reactive group (e.g., an alkyne reactive group) can comprise a structure of formula (E-1'):
wherein J is a reactive group, (e.g., an alkyne reactive group; e.g., azide, diene, nitrone, or nitrile oxide); and y is an integer from 0 to 20 (e.g., 9).
The alkyne reactive group containing compound can comprise the structure of formula (E-1):
where y is an integer from 0 to 20 (e.g., 9).
Reacting an alkyne moiety (e.g., an alkyne covalently attached to a nanoparticle) with a compound comprising an alkyne reactive group (e.g., a compound of formula D, such as D-1) can provide a compound of formula (NP-2):
where x is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, e.g., 4), and y is an integer from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, such as 3), and the silicon atom is part of the nanoparticle (e.g., bonded to the silica shell of the core-shell silica nanoparticle).
Reacting an alkyne moiety (e.g., an alkyne covalently attached to a nanoparticle) with a compound comprising an alkyne reactive group (e.g., a compound of formula E, such as E-1) can provide a compound of formula (NP-3):
where x is an integer from 0 to 10 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, e.g., 4), and y is an integer from 0 to 20 (e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, e.g., 9), and the silicon atom is part of a nanoparticle (e.g., bonded to the silica shell of a core-shell silica nanoparticle).
An exemplary NDC comprising a payload (isatecan) linked by a protease cleavable linker, and a mechanism for linker cleavage and payload release are described in scheme 2.
Scheme 2. Exemplary NDCs produced by the methods disclosed herein and cleavage thereof by cathepsin B (Cat-B).
The methods disclosed herein can be used to obtain nanoparticle drug conjugates having a desired number of targeting ligands or payload moieties. For example, NDCs produced using the methods disclosed herein may have nanoparticle to payload ratios of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, 1:30, 1:32, 1:34, 1:36, 1:38, 1:40, 1:45, 1:50, 1:55, 1:60, 1:65, 1:70, 1:75, or 1:80. For example, the average number of ixabepilone molecules per nanoparticle may be between about 5 and about 10, between about 10 and about 15, between about 15 and about 20, between about 20 and about 25, or between about 25 and about 30, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 ixabepilone molecules per nanoparticle. NDCs produced using the methods disclosed herein can have an average nanoparticle to targeting ligand (e.g., folic acid) ratio of about 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, 1:25, 1:26, 1:27, 1:28, 1:29, or 1:30. For example, the average nanoparticle to targeting ligand ratio may be in the range of about 1 to about 20, e.g., the average number of folate molecules per nanoparticle may be between about 5 to about 10, between about 10 to about 15, or between about 15 to about 20, e.g., about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, or about 15 folate molecules per nanoparticle.
It should be understood that while the methods exemplified herein are characterized by functionalizing the nanoparticle with alkynes, and conjugating compounds comprising alkyne reactive moieties to the nanoparticle, these functionalizations are vice versa. For example, the methods disclosed herein can be modified to functionalize nanoparticles with alkyne reactive moieties (e.g., azide, diene, nitrone, or nitrile oxide), and further conjugate the functionalized nanoparticles to alkyne-containing compounds. For example, the nanoparticle may be functionalized with a first bifunctional precursor and then conjugated to a second bifunctional precursor comprising an alkyne reactive group, thereby providing a nanoparticle functionalized with an alkyne reactive group (e.g., azide, diene, nitrone, or nitrile oxide). Then, the alkyne-containing compound (e.g., the targeting ligand or payload) can be reacted with the nanoparticle to conjugate the compound to the nanoparticle.
The methods disclosed herein can overcome the shortcomings of other methods of functionalizing nanoparticles. For example, the methods disclosed herein can address the limited stability of nanoparticle conjugates prepared using other methods. In particular, the methods disclosed herein exhibit relatively stable bifunctional precursors that do not promote hydrolysis of bonds in the linker moiety, which may occur when using bifunctional precursors comprising highly reactive groups (e.g., certain amine groups). Furthermore, the methods disclosed herein employ bifunctional precursors that do not substantially self-condense during the reaction, which may occur when other bifunctional precursors (e.g., amine-containing bifunctional precursors) are used, for example, due to electrostatic attraction or other undesirable intermolecular or intramolecular interactions. Overcoming these problems can greatly increase yields and avoid difficult or impossible purification procedures that would otherwise be necessary, which is particularly important for generating NDC for clinical use. Thus, the methods disclosed herein are particularly useful for producing NDCs intended for clinical applications due to the simplicity and reliability in producing high purity functionalized nanoparticles.
Functionalized nanoparticles produced using the methods disclosed herein can exhibit uniform morphology and narrow size distribution, for example, as measured by Transmission Electron Microscopy (TEM). The nanoparticles may be characterized by TEM or UV spectroscopy, for example, to identify absorption peak characteristics of the dye or ligand in or on the nanoparticle. The purity of the nanoparticles can be assessed using chromatography, for example, reverse phase high performance liquid chromatography (RP-HPLC) and size exclusion chromatography. The methods disclosed herein are also capable of providing highly consistent nanoparticle characteristics across a variety of reaction scales and batches.
Examples
The following examples are provided in order that the invention described herein may be more fully understood. These examples are provided to illustrate the methods and functionalized nanoparticles provided herein and should not be construed as limiting their scope in any way.
The compounds provided herein can be prepared from readily available starting materials using modifications to the specific synthetic schemes listed below that are well known to those skilled in the art. It should be understood that other process conditions may be used where typical or preferred process conditions (i.e., reaction temperature, time, molar ratio of reactants, solvents, pressures, etc.) are given unless otherwise indicated. The optimum reaction conditions may vary with the particular reactants or solvents used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
Furthermore, it will be apparent to those skilled in the art that conventional protecting groups may be required to prevent unwanted reactions of certain functional groups. The selection of suitable protecting groups for a particular functional group and the appropriate conditions for protection and deprotection are well known in the art. For example, greene et al, protecting Groups in Organic Synthesis, second Edition, wiley, new York,1991 and references cited therein describe various protecting groups, and their introduction and removal.
Materials and methods
All materials were used as received. All non-aqueous reactions were performed in flame-dried glassware under positive argon pressure. Anhydrous solvents were purchased from commercial suppliers (RANKEM). Flash chromatography was performed on 230-400 mesh silica gel using the indicated solvent system. Proton nuclear magnetic resonance spectra were recorded on a Bruker spectrometer using deuterated chloroform or DMSO-acting solvents at 400 MHZ. The peak positions are given in parts per million starting from the low field of tetramethylsilane as internal standard. J values are expressed in hertz. Mass spectrometry was performed on an (Agilent/Shimadzu) spectrometer using Electrospray (ES) technique. HPLC analysis was performed on an (Agilent/Waters) PDA-UV detector equipped with Gemini C-18 (1000X4.6 mm;5 u) and using this method to determine the purity of all compounds tested was >95%. Compounds prepared according to the methods described herein may be isolated by preparative HPLC methods. Cyanine 5maleimide (Cyanine 5 maleimide) was purchased from Lumiprobe. DBCO-PEG 4-maleimide, folic acid-PEG-azide and irinotecan-linker conjugates were obtained from the client synthesis of the free drug Mingkang (Tianjin, china). (3-mercaptopropyl) trimethoxysilane, 2- [ methoxy (polyethyleneoxy) 6-9 propyl ] trimethoxysilane, and (3-cyclopentadienyl propyl) triethoxysilane precursors are purchased from Gelest.10x PBS was purchased from Thermo Fisher Scientific. Human cathepsin B was purchased from Millipore Sigma. Human and mouse serum was purchased from BIOIVT (NewYork). All other chemicals were purchased from Millipore Sigma. Cy5-C' Dot was prepared according to the protocol described in Ma, K.et al, chemistry of Materials (2015) 27 (11): 4119-4133, which is incorporated herein by reference in its entirety.
Gel Permeation Chromatography (GPC) characterization and purification was performed according to the procedure outlined in Chen, F. Et al, chemistry of Materials (2017) 29 (20): 8766-8779. Transmission Electron Microscopy (TEM) characterization was performed using Titan Cubed Themis TEM and High Angle Annular Dark Field (HAADF) imaging to confirm the nanoparticle distribution. UV-visible spectral analysis of the samples was performed using a Cary 5000UV-Vis-NIR spectrophotometer. Fluorescence Correlation Spectroscopy (FCS) measurements were performed using a self-contained FCS apparatus as described in Ma, K.et al, chemistry of Materials (2016) 28 (5): 1537-1545. Cy5 dye encapsulated in the silica core of C' Dot was excited using a 633nm solid state laser. The purity of nanoparticle conjugates (including FA-CDC) was assessed using RP-HPLC coupled with a photodiode array (PDA) detector using a commercial Waters Xbridge Peptide BEH C18 column (50 x 4.6 mm). The mobile phase used for separation was eluted by a gradient starting from a composition of 85% a (0.1 vol%, trifluoroacetic acid in deionized water)/15% B (acetonitrile) and the composition was linearly changed to 5%A/95% B over 15 minutes. RP-HPLC separates molecules with different polarities and is suitable as an analytical method for FA-CDC (due to its ultra-small sub-10 nm particle size). Nanoparticles and other chemical moieties (e.g., ligands that are non-covalently bound to the nanoparticles and the degrading or force-releasing components) can be well separated using RP-HPLC. Different chemical moieties were identified based on elution time and unique UV/visible spectra collected using PDA detectors. The impurity content in the sample can be quantified using a calibration curve developed using a reference standard material for the key impurities. Size Exclusion Chromatography (SEC) characterization was performed using a Tosoh Bioscience PWXL 4000 7.8mm x 300mm column coupled to a PDA detector with a mobile phase consisting of 80% sodium chloride in 0.9wt.% deionized water and 20% acetonitrile. Separation occurs in isocratic mode over the course of 30 minutes, and a typical flow rate for separation is 0.6mL/min.
Example 1. Synthesis of exemplary functionalized nanoparticles using DBCO-PEG-silane.
Scheme 3. Exemplary one-step method for functionalizing nanoparticles with DBCO using DBCO-PEG-silane.
In a round bottom flask with a stir bar, cy5-C' Dot is diluted to the desired concentration, typically between 15 and 30. Mu.M, with deionized water. DBCO-PEGn-silane (n=4 or 12) was dissolved in DMSO and added to the reaction with stirring to achieve the desired particle to DBCO molar ratio. After reaction at room temperature overnight, the reaction solution was concentrated and purified using Gel Permeation Chromatography (GPC). The number of DBCO groups attached to the nanoparticle is provided in fig. 2.
Example 2. Synthesis of exemplary functionalized nanoparticles using amine-based precursors.
Silanol groups on the nanoparticle surface under an organic polymer coating (e.g., PEG layer) are not readily accessible to macromolecules, such as DBCO-PEG-silane. As a result, when DBCO-PEG-silane is used as a bifunctional precursor (e.g., as in example 1), only one or two DBCO groups can be attached to each nanoparticle (e.g., C' Dot) even at high reaction ratios of DBCO-PEG-silane to nanoparticles (see fig. 2). An alternative two-step reaction was therefore devised, employing relatively small silane molecules with high diffusion coefficients, as shown in scheme 4.
Scheme 4. Exemplary two-step method of functionalizing nanoparticles with DBCO using amine-silane bifunctional precursors. In a round bottom flask with a stir bar, cy5-C' Dot is diluted to the desired concentration, typically between 15 and 30. Mu.M, with deionized water. (3-aminopropyl) triethoxysilane (APTMS) was added to the C' Dot solution with stirring to achieve the desired molar ratio of particles to APTMS. After overnight reaction, DBCO-PEG4-NHS ester in DMSO was added to achieve the desired particle to DBCO molar ratio and further conjugated to amine groups on C' Dot via amine-ester reaction. The reaction was left at room temperature overnight, then concentrated and purified using GPC. After GPC purification, the purified particles in 0.9% normal saline were heated to 80 ℃ overnight in a water bath. After heat treatment, the solution was concentrated and purified again using GPC to obtain amine-based DBCO-C' Dot. This two-step reaction utilizes rapid diffusion of silane small molecules to enhance their reactivity with silanol groups under the PEG layer, allowing for easy attachment of more than 20 DBCO groups to each C' Dot (see fig. 2) as compared to a one-step reaction.
However, amine-based DBCO-C' Dot produced using this approach exhibits limited stability. For example, after overnight incubation at 37 ℃ in PBS, a significant amount of DBCO is detached from the C ' Dot (see fig. 3A), which may be caused by the accelerated hydrolysis of the amide bond between the DBCO groups and the C ' Dot by the residual primary amine groups on the C ' Dot surface. In addition, amine-silane molecules can self-condense during the reaction due to electrostatic attraction between positively charged amine moieties and negatively charged silanol moieties. Amine-silane aggregates are difficult to remove from the C' Dot solution by Size Exclusion Chromatography (SEC) (see fig. 3C) and can cause undesirable downstream effects. For example, if administered, these aggregates may lead to renal retention due to the positive charge of the amine.
EXAMPLE 3 screening for conjugate chemical Synthesis
To overcome the shortcomings of using amine-silane precursors described in example 2, a chemical library of conjugates was screened and summarized in table 1.
Table 1. Chemical screening of conjugates.
a The number of azide groups per particle cannot be quantified directly from the UV-Vis spectrum. Thus, the DBCO-Cy3 molecule is further conjugated to an azide functionalized C' Dot, attaching Cy3 to the particle by a click chemistry reaction between DBCO and azide; b at 290nm, the presence of alkyne groups on C' dot is verified by the specific absorbance peak of alkyne; c the synthesis scheme is optimized to obtain; d the DBCO-Cy3 molecule was further conjugated to an azide functionalized C' Dot, and Cy3 was attached to the particle by a click chemistry reaction between DBCO and azide.
EXAMPLE 4 diene-based DBCO-C' Dot Synthesis
In example 3, the use of Diels-Alder reactions was identified as a suitable alternative to amine-ester reactions. For example, the amine-silane used in the process described in example 2 may be replaced with (3-cyclopentadienyl) triethoxysilane ("diene-silane") to functionalize the C' Dot with cyclopentadienyl groups first, and the DBCO-PEG-NHS ester used in the process of example 2 may be replaced with DBCO-PEG-maleimide. This method is depicted in scheme 5, wherein a Diels-Alder reaction between a maleimide group on DBCO-PEG-maleimide and a diene group on C' Dot can be used to attach DBCO (via a linker) to the nanoparticle.
Scheme 5. Exemplary two-step process for functionalizing nanoparticles with DBCO using a diene-silane bifunctional precursor.
In a round bottom flask with a stir bar, cy5-C' Dot is diluted to the desired concentration, typically between 15 and 30. Mu.M, with deionized water. (3-cyclopentadienyl) triethoxysilane (cyclopentadiene) was first diluted 100x in DMSO and then added to the reaction with stirring to achieve the desired molar ratio of particles to cyclopentadiene. After overnight reaction, 10x PBS was added to the reaction to reach the final concentration of 1x PBS. Next, DBCO-maleimide precursor (DBCO-maleimide, DBCO-PEG 4-maleimide, DBCO-PEG 12-maleimide or DBCO-sulphonated-maleimide) is dissolved in DMSO and added to the reaction to achieve the desired particle to DBCO molar ratio. After mixing for about 30 minutes to 1 hour, the reaction mixture was heated to 80 ℃ while stirring overnight. The reaction solution was then concentrated and purified using Gel Permeation Chromatography (GPC) to obtain diene-based DBCO-C' Dot.
Without wishing to be bound by theory, it is believed that the neutral charge of the cyclopentadienyl group avoids the hydrolysis of the amide bond in the bond, accelerated by the primary amine on C' Dot, when an amine-silane is used as a precursor. Thus, the C' Dot generated using this method is highly stable (see fig. 3B). In addition, the use of cyclopentadienyl groups greatly reduces self-condensation of silane during the reaction (see fig. 3D) and improves stability, size uniformity, reaction yield and purity of functionalized nanoparticles (e.g., DBCO-C' Dot).
The reaction efficiency of a DBCO-containing precursor (e.g., DBCO-maleimide) is independent of its diffusion coefficient. For example, even when DBCO-maleimide precursors having different PEG spacer lengths and molecular weights are applied to functionalized C 'Dot, the number of DBCO groups attached to each resulting C' Dot remains consistent (see fig. 2). This is in contrast to typical processes involving other bifunctional precursors, where the reaction efficiency is greatly affected by the molar mass of the silane. This also shows that the low yield of C 'Dot reaction with bulky silanes is mainly due to the self-condensation of silanes, which competes with their diffusion into the nanoparticle organic polymer coating (e.g., C' Dot PEG layer) and subsequent reaction. The reaction between the DBCO-maleimide precursor and the nanoparticle (e.g., C' Dot) may be sensitive to the hydrophobicity and charge state of the DBCO-maleimide precursor used. For example, relatively hydrophilic molecules having a negative charge (e.g., DBCO-sulfonated-maleimide with hydrophilic sulfonated spacers) exhibit relatively poor reactivity. Without wishing to be bound by theory, this poor reactivity may be due to increased water solubility and electrostatic repulsion of negatively charged silanol groups. Based on these results, a short PEG spacer with 4 ethylene glycol units was used in the DBCO-maleimide precursor to give the desired accessibility for subsequent modification of the DBCO groups on the C' Dot surface (e.g., using click chemistry).
EXAMPLE 5 Synthesis of FA-CDC
NDCs comprising alkyne moieties (e.g., DBCO) produced by the methods disclosed herein can be further conjugated with compounds comprising reactive groups (e.g., alkyne reactive groups (e.g., azides)), which can include payload-linker conjugate precursors, e.g., as described in synthesis example 1, and/or folic acid targeting ligand precursors, e.g., as described in synthesis example 2.
For example, using click chemistry, DBCO-C' Dot produced by the methods disclosed herein is conjugated with an azide-functionalized folate-linker conjugate (suitable as a targeting ligand for targeting a Folate Receptor (FR) expressing cancer) and an azide-functionalized isatecan-linker conjugate (isatecan is a topoisomerase I inhibitor drug that is useful for treating cancer). The folate moiety is linked by a non-cleavable linker, while the isatecan moiety is attached by a cathepsin B (Cat-B), an enzyme that is present in the lysosome and is overexpressed in a variety of malignancies. These methods are depicted in scheme 6.
Scheme 6. Exemplary method of functionalizing nanoparticles with folic acid fraction and ixabepilone fraction.
In a round bottom flask with a stir bar, DBCO-C' Dot is diluted with deionized water to the desired concentration, typically between 15 and 30. Mu.M. To functionalize DBCO-C' Dot with folic acid ligand, folic acid-PEG-azide was dissolved in DMSO and added to the reaction with stirring to achieve the desired amount of ligand per particle. The reaction was left at room temperature overnight and the conversion (typically greater than 95%) was checked by in-process HPLC purity testing. If the purity is less than 95%, the reaction solution is concentrated and purified using GPC. Folate-functionalized C' Dot was isolated as an intermediate to determine the amount of folate ligand per particle using UV-Vis. To further functionalize folic acid in C' Dot with cleavable ixatin Kang Zhuige, particle concentration was adjusted as needed. The cleavable linker-irinotecan conjugate was dissolved in DMSO and added to the particle solution with stirring to achieve the desired amount of payload per particle. The reaction was left at room temperature overnight, then the reaction solution was concentrated and purified using GPC to obtain FA-CDC.
The resulting targeted NDCs exhibited uniform morphology and narrow size distribution under Transmission Electron Microscopy (TEM), with an average core size of 3.9 nm. NDC has an average hydrodynamic diameter of 6.4nm (fig. 4) as determined by Fluorescence Correlation Spectroscopy (FCS), consistent with TEM, as the 2.5nm difference is due to the organic PEG layer that is not observable in TEM. The final purified NDC had a purity of greater than 99.0% as characterized by reverse phase high performance liquid chromatography (RP-HPLC) and SEC (fig. 6A-6B). These quality attributes of NDC were demonstrated to be highly consistent between batches of different reaction scales (table 2).
Table 2. Characteristics of NDC between different batches on an exemplary scale.
When C' Dot was sequentially functionalized with cyclopentadiene, DBCO, folic acid, and ixatikang to produce targeted NDC, UV-Vis spectra were collected at each step. The characteristic absorption peaks were used to verify the presence of each ligand and determine the number of ligands per particle (fig. 5). DBCO-C' Dot shows an absorption peak at 647nm, corresponding to covalently encapsulated Cy5; bimodal at 308nm and 290nm, corresponding to DBCO groups, was shown. The molar concentrations of Cy5 and DBCO can be determined by applying Beer-Lambert law at 647nm and 308nm, respectively, using their extinction coefficients. The particle concentration was obtained by dividing the molar concentration of Cy5 by the number of Cy5 dyes per particle determined by FCS. The number of DBCO groups per particle is obtained by dividing the molar concentration of DBCO groups by the particle concentration.
Before attaching the isartan payload, the UV-Vis spectrum of the folate-functionalized C' Dot was used to determine the concentration of particles and the concentration of FR targeting ligand by applying Beer-Lambert law at 647nm (corresponding to covalently encapsulated Cy 5) and 360nm (corresponding to folate targeting ligand), respectively. The number of FR-targeting ligands per particle is determined by dividing the molar concentration of FR-targeting ligand by the particle concentration. The number of FR-targeting ligands has been shown to remain unchanged during the subsequent course of FA-CDC production.
Similarly, the UV-Vis spectrum of FA-CDC can be used to determine the concentration of FA-CDC particles and the concentration of cleavable irinotecan at 647nm and 360nm (corresponding to folate targeting ligand and irinotecan) by subtracting the absorbance of the FR targeting ligand at 360 nm. The concentration of irinotecan was then divided by the concentration of FA-CDC particles to determine the amount of irinotecan per particle.
EXAMPLE 6 stability of NDC in physiological saline, PBS, human and mouse serum
The stability of NDCs produced herein was evaluated after incubation in shaking dry baths for different periods of time at 37 ℃ in 0.9% physiological saline, PBS, human plasma (10%) and mouse plasma (10%). Prior to analysis, plasma proteins in the samples were removed by adding an equal volume of cold acetonitrile pellet, and then centrifuged at 10000rpm in an Eppendorf 5425 microcentrifuge. After centrifugation, the clarified supernatant was transferred from the centrifuge tube to a clear total recovery HPLC vial. The supernatant, free of any visible aggregates, was diluted with an equal volume of deionized water to adjust the sample matrix to match the starting conditions for HPLC separation and avoid sensitivity loss. The purity and impurities of each sample were then quantified by RP-HPLC as described above. The stability of NDCs produced by the methods of example 2 and example 4 in human and mouse serum is provided in fig. 8A-8B.
Nanoparticle conjugate samples (e.g., FA-CDC) were prepared for forced release by first diluting an aliquot of the FA-NDC sample to a concentration of 2 μm and then incubating with activated recombinant human cathepsin-B in a shaking dry bath at 37 ℃. The preparation process of the recombinant human cathepsin-B is as follows: mu.L of 0.33. Mu.g/. Mu.L of cathepsin-B was added to an activation buffer consisting of dithiothreitol and (MES) and adjusted to pH 5.0. After preparation, the appropriate pH was confirmed using pH paper. After 24 hours of incubation, the force to release free irinotecan was determined by the percentage of the integrated area present over the elution time of free irinotecan in RP-HPLC. The results of this study are provided in fig. 7.
When the targeted NDC was incubated with recombinant human Cat-B (SI), more than 95% of the irinotecan drug was released from NDC within 48 hours, with a fitting half-life of around 3.0 hours (fig. 7), confirming efficient drug release in the cancer environment. Targeted NDCs produced using diene-silane precursors (as depicted in scheme 5) also exhibited high stability in mouse and human plasma, and improved stability relative to their counterparts produced using amine-silanes (as depicted in scheme 4) (fig. 8A and 8B). It was found by UV-Vis spectroscopy of the NDC peak in RP-HPLC chromatograms that over 95% of the irinotecan drug remained on NDC for up to 7 days in mouse and human plasma. At the same time, monitoring an independent RP-HPLC assay for free irinotecan indicated that released irinotecan was below the detection limit of RP-HPLC, i.e., 0.02%, and the absence of unwanted free drug further confirmed their high plasma stability. The targeted NDCs also exhibit high storage stability in 0.9% physiological saline at 4 ℃. Their purity, particle size distribution and hydrodynamic diameter were characterized by RP-HPLC, SEC and FCS, respectively, and remained unchanged for more than 6 months under storage conditions. Such high storage stability is another key parameter important for both clinical transformation and commercial production.
Synthesis example 1: synthesis of linker-payload conjugate precursors
Linker-payload conjugate precursors suitable for use in the methods disclosed herein may be synthesized according to the following exemplary schemes.
(S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p) Tolyl) methyl) amino) hexanamide(161) Is synthesized by (a)
Scheme 7: synthesis of Compound (161).
Synthesis of 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159): imidazole (5.54 g,81.22 mmol) was added to a solution of (4-aminophenyl) methanol (75) (5.0 g,40.61 mmol) in DMF (25 mL) at 0deg.C, then tert-butyl (chloro) diphenylsilane (13.39 g,48.73 mmol) was added and the reaction mixture was stirred at room temperature for 16h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was quenched with water (20 mL) and extracted with EtOAc (2×200 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 Dried, concentrated under reduced pressure, and purified by column chromatography on silica gel (230-400 mesh) eluting with 10% EtOAc in petroleum ether to afford 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159, 6.6 g) as a gum. LCMS: M/z 362.31[ (M+H) + ];R t 2.58min;93.68% purity.
Synthesis of (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160): diisopropylethylamine (4.18 mL,24 mmol), HATU (6.08 g,16 mmol) and 4- (((tert-butyldiphenylsilyl) oxy) methyl) aniline (159) (2.89 g,8 mmol) were added to a solution of N2- (((9H-fluoren-9-yl) methoxy) carbonyl) -N6- (diphenyl (p-tolyl) methyl) -L-lysine (149) (5.0 g,8 mmol) in DMF (50 mL) at 0deg.C and the reaction mixture stirred at room temperature for 16H. Through TLC monitored the progress of the reaction. After completion of the starting material, the reaction mixture was quenched with ice water. The precipitated solid was filtered and dried under vacuum to provide (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160; 5.5 g) as a solid. LCMS: M/z 990.37[ (M+H) + ];R t 2.84min;96.79% purity.
Synthesis of (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (161): piperidine (16.5 mL) was added to a solution of (9H-fluoren-9-yl) methyl (S) - (1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) carbamate (160) (5.5 g,5.68 mmol) in DMF (38.5 mL) at room temperature and the reaction mixture was stirred at room temperature for 3H. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by column chromatography using silica gel (230-400 mesh) eluting with 10% EtOAc to provide (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (160; 3.5 g) as a gum. LCMS: m/z744.24[ (MH) - ];R t 2.20min;90.16% purity.
4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl
Phenyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-amide
Synthesis of (phenyl) benzyl (4-nitrophenyl) carbonate (191)
Scheme 8: synthesis of Compound (191).
(9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6-Synthesis of (diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl amino) -3-methyl-1-oxobutan-2-yl carbamate (187): diisopropylethylamine (1.54 mL,8.83 mmol), HATU (2.24 g,5.89 mmol) and (S) -2-amino-N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (161) (2.19 g,2.94 mmol) were added to a solution of (((N- (9-fluorenylmethoxycarbonyl) -L-valine (1 g,2.94 mmol) in DMF (20 mL) and the reaction mixture was stirred at room temperature for 3H. After completion of the starting material by TLC monitoring the reaction mixture, the precipitated solid was filtered and dried under vacuum to provide (((9H-fluoren-9-yl) methyl ((S) -1- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxo-2-hexane-2-oxo-2-methyl) amino) butan-2-yl ester which was 5g of (LCM-2-oxo-2-amino-butan-2-yl ester + 1067, retention time 2.42min.
Synthesis of (S) -2- ((S) -2-amino-3-methylbutanamido) -N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) acetamide (188): a30% piperidine solution in DMF (4.5 mL) was added to a solution of (((9H-fluoren-9-yl) methyl ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) carbamate (187) (1.5 g,1.40 mmol) in DMF (6 mL) at room temperature and the reaction mixture was stirred at room temperature for 2H. After completion of the starting material, the reaction mixture was concentrated under reduced pressure and purified by flash chromatography eluting with 100% EtOAc to provide (S) -2- ((S) -2-amino-3-methylbutanoylamino) -N- (4- ((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (188) as a solid, 1 g. 1 H NMR(400MHz,DMSO-d 6 ):δ10.07(s,1H),7.64-7.63(d,4H),7.56-7.54(d,2H),7.46-7.35(m,9H),7.27-7.24(m,8H),7.185-7.11(m,2H),7.05-7.03(d,2H),4.71(s,2H),4.44(d,1H),3.25-3.16(d,1H),3.01-3.00(m,1H),2.21(s,3H),1.98-1.93(m,2H),1.68-1.38(m,4H),1.15(s,10H),LCMS:MH + 845, retention time 3.63min.
Synthesis of 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (189): diisopropylethylamine (0.49 mL,2.83 mmol), HATU (719.47 mg,1.89 mmol) and (S) -2- ((S) -2-amino-3-methylbutanoylamino) -N- (4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) -6- ((diphenyl (p-tolyl) methyl) amino) hexanamide (188) (800 mg,0.94 mmol) were added to a solution of 1-azido-3,6,9,12,15,18,21,24,27-nonaoxatriacontane-30-oic acid (86) (254 mg,0.94 mmol) in DMF (8 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was quenched with water (15 mL) and extracted with EtOAc (2×30 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 Dried, concentrated under reduced pressure, and purified by flash chromatography eluting with 3% MeOH in DCM to provide 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (189; 0.60 g) as a gum. 1 H NMR(400MHz,DMSO-d 6 ):δ9.91(s,1H),8.02-8.00(d,2H),7.95(s,2H),7.87-7.85(d,1H),7.64-7.63(d,4H),7.57-7.55(d,2H),7.46-7.32(m,11H),7.26-7.24(m,8H),7.15-7.11(t,2H),7.05-7.03(d,2H),4.71(s,2H),4.35-4.33(m,1H),4.19(s,1H),3.59-3.36(m,38H),2.68-2.38(m,6H),2.22(s,3H),1.98-1.92(m,2H),1.47-1.17(m,4H),1.02(s,9H),0.85-0.80(m,6H).LCMS:MH + 1338, retention time 2.92min.
Synthesis of 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (190): at room temperatureNext, NH 4 F (166 mg,4.48 mmol) was added to a solution of 1-azido-N- ((S) -1- (((S) -1- ((4- (((tert-butyldiphenylsilyl) oxy) methyl) phenyl) amino) -6- ((diphenyl (p-tolyl) methyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (189) (600 mg,0.44 mmol) in methanol (10 mL) and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting material, the reaction mixture was concentrated under reduced pressure, and the obtained residue was diluted with water (15 mL) and extracted with EtOAc (2×20 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 Dried, concentrated under reduced pressure, and purified by flash chromatography eluting with 5% MeOH in DCM to provide 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (190; 0.40 g) as a gum. 1 H NMR(400MHz,DMSO-d 6 ):δ9.81(s,1H),7.96-7.94(d,1H),7.84-7.81(d,1H),7.53-7.51(d,2H),7.37-7.35(d,4H),7.26-7.12(m,9H),7.096-7.04(d,2H),5.06-5.04(t,1H),4.43-4.41(d,2H),4.35(m,1H),4.18-4.16(t,1H),3.60-3.46(m,33H),3.39-3.36(t,2H),2.50-2.23(m,2H),2.23(s,3H),2.23-1.93(m,2H),1.48-1.23(m,6H),0.85-0.80(m,6H).LCMS:MH + 1100, retention time 3.72min.
Synthesis of 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl (4-nitrophenyl) carbonate (191): pyridine (0.14 mL,1.80 mmol) and nitrophenyl 4-chloroformate (14) (145 mg,0.72 mmol) were added to a solution of 1-azido-N- ((S) -1- (((S) -6- ((diphenyl (p-tolyl) methyl) amino) -1- ((4- (hydroxymethyl) phenyl) amino) -1-oxohexan-2-yl) amino) -3-methyl-1-oxobutan-2-yl) -3,6,9,12,15,18,21,24,27-nonaoxatriacontan-30-amide (190) (400 mg,0.36 mmol) in DCM (10 mL) at 0deg.C and the reaction mixture was stirred at room temperature for 6h. The progress of the reaction was monitored by TLC. After completion of the starting materials, the reaction mixture is Concentrated under reduced pressure and purified by flash chromatography eluting with 3% MeOH in DCM to give 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl (4-nitrophenyl) carbonate (191; 0.34 g) as a gum. LCMS: MH + 1265, retention time 1.33min.
4- ((32S, 35S) -35- (4-aminobutyl) -1-azido-32-isopropyl-30,33-dioxo-3, 6,9,
12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl
1H, 12H-benzo [ de ] carbonyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H]Pyrano-s
[3’,4’:6,7]Indolo [1,2-b ]]Synthesis of quinolin-1-yl) carbamate (202).
Scheme 9: protease may cleave the synthesis of linker-payload conjugate precursors (202).
4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] ]Pyrano [3',4':6,7]Indolo [1,2-b ]]Synthesis of quinolin-1-yl) carbamate (201): triethylamine (0.09 mL,0.62 mmol) and (1R, 9R) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ de ] at 0deg.C]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinoline-10, 13-Diketomethanesulfonate (i Sha Tikang methanesulfonate; 16;131mg,0.25 mmol) 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl (4-nitrophenyl) carbonate in NMP (2.5 mL)(191; 311mg,0.25 mmol) and the reaction mixture was stirred at room temperature for 8h. The progress of the reaction was monitored by LCMS. After completion of the starting material, the reaction mixture was quenched with water (15 mL) and extracted with 10% methanol in chloroform (2×20 mL). The combined organic layers were dried over anhydrous sodium sulfate (Na 2 SO 4 ) Drying and concentrating under reduced pressure. Diethyl ether was added to the crude product and the resulting precipitate was filtered and purified using column chromatography (Combi-Flash) eluting with 5% MeOH in DCM to provide 4- ((32 s,35 s) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-amido) benzyl ((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1 h,12 h-benzo [ de ] ]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl) carbamate (201) as a solid (0.3 g). LCMS: MH + 1561, retention time 2.18min.
4- ((32S, 35S) -35- (4-aminobutyl) -1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3',4':6,7]Indolo [1,2-b ]]Synthesis of quinolin-1-yl) carbamate (202): a1% solution of trifluoroacetic acid (TFA) in DCM was added to 4- ((32S, 35S) -1-azido-35- (4- ((diphenyl (p-tolyl) methyl) amino) butyl) -32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-acylamino) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ] in DCM (5 mL) at 0deg.C]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl) carbamate (201; 300mg,0.19 mmol) and the reaction mixture was stirred at room temperature for 1h. The progress of the reaction was monitored by LCMS. After completion of the starting material, the reaction mixture was concentrated under reduced pressure, the residue was triturated with diethyl ether and purified by RP-prep-HPLC to give 4- ((32S, 35S) -35- (4-aminobutyl) -1-azido-32-isopropyl-30,33-dioxo-3,6,9,12,15,18,21,24,27-nonaoxa-31, 34-diazatricetyl-36-amido) benzyl ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dioxo-2,3,9,10,13,15-hexahydro-1H, 12H-benzo [ de ]]Pyrano [3',4':6,7]Indolo [1,2-b ]]Quinolin-1-yl) carbamate (202) (70 mg) as a solid. 1 H NMR(400MHz,DMSO-d 6 ):δ9.96(s,1H),8.12-8.10(q,2H),7.89-7.87(d,1H),7.76-7.61(d,1H),7.59-7.31(m,7H),6.51(s,1H),5.44(s,2H),5.29(s,3H),5.09(s,2H),4.37-4.20(m,1H),4.18-4.16(t,1H),3.49-3.44(m,4H),3.12-2.55(m,39H),2.40-1.34(m,15H),0.89-0.82(m,9H),LCMS:MH + 1305, retention times 5.33 and 5.47min.
Synthesis example 2: synthesis of targeting ligand precursors
Targeting ligand precursors suitable for use in the methods disclosed herein can be prepared according to the following exemplary synthetic schemes.
(S) -16- (4- (((2-amino-4-oxo-3, 4-dihydropteridin-6-yl) methyl) amino) benzoylamino) -1-
Synthesis of azido-13-oxo-3, 6, 9-trioxa-12-aza-heptadecane-17-acid (606).
Scheme 10: synthesis of folate receptor targeting ligand (606).
Preparation of Compound 600: compound 599 (160 g,512 mmol) was dissolved in TFAA (800 mL) at 25℃and stirred in the dark under nitrogen for 5h. The solvent was then removed in vacuo at 50 ℃ to give the crude product. The crude product was triturated with MTBE (750 mL) for 60min and then filtered to provide compound 600 (203 g, crude) as a solid which was used in the next step without further purification. LC-MS: 1 H NMR:(400MHz,CDCl 3 )δ12.74(br s,1H),8.88(s,1H),7.97-8.05(m,2H),7.66-7.74(m,2H),5.26(s,1H)。
preparation of Compound 602: TBTU (238 g,740 mmol) and DIPEA (95.7 g,740 mmol) were added to a solution of compound 601 (225 g,529 mmol) in DMF (2.25L). Stirring at 20 ℃ for 30min, adding 2- (2- (2- (2-azidoethoxy) ethoxy) ethan-1-amine (reagent a; 121g, 55mmol) and the mixture was stirred at 50℃for 12 hours. The two reaction mixtures were combined and worked up by H 2 The residue was diluted with O (3L) and extracted with ethyl acetate (1500 mL. Times.3). The combined organic layers were washed with brine (800 ml x 3), taken up in Na 2 SO 4 Dried, filtered and concentrated under reduced pressure, purified by column chromatography (SiO 2 Petroleum ether/ethyl acetate=100/1 to 1/1) to afford compound 602 (590 g) as an oil. 1 H NMR:(400MHz,CDCl 3 )δ7.76-7.78(m,2H),7.63-7.60(m,2H),7.41-7.27(m,4H),6.43(s,1H),5.70(s,1H),4.42-4.38(m,2H),4.24-4.23(m,2H),3.63-3.36(m,16H),2.28-2.18(m,3H),1.98-1.96(m,1H),1.48(s,9H)。
Preparation of Compound 603: n-ethyl ethylamine (1.27 kg,17.4 mol) was added to a solution of compound 602 (435 g,695 mmol) in DCM (4.35L) and the mixture stirred at 25℃for 3 h. The solvent was then removed in vacuo at room temperature and the residue purified by flash column chromatography (DCM/meoh=100/1 to 1/1) to afford compound 603 (245 g), 1 H NMR:(400MHz,CDCl 3 )δ6.55(s,1H),3.67-3.30(m,17H),2.34-2.30(m,2H),2.10-2.06(m,1H),1.87(s,2H),1.77-1.73(m,1H),1.44(s,9H)。
preparation of compound 604: TBTU (119 g, 178 mmol) and DIEA (160 g,1.24 mol) were added to a solution of compound 600 (101 g,248 mmol) in DMF (900 mL) and the mixture stirred for 30 min. Compound 603 (100 g,248 mmol) in DMF (100 mL) was added. The mixture was stirred at 25℃for 12 hours. The two reaction mixtures were combined and concentrated using H 2 The residue was diluted with O (2.5L) and extracted with ethyl acetate (1L x 5). The combined organic layers were washed with brine (600 ml x 3), taken up in Na 2 SO 4 Dried, filtered and concentrated under reduced pressure to afford compound 4 (420 g, crude) as a solid, which was used in the next step without further purification.
Preparation of Compound 605: will K 2 CO 3 (585 g,4.23 mol) to THF (4.2 mL) and H 2 A solution of compound 604 (420 g,529 mmol) in O (500 mL) was stirred at 60℃for 0.5 h. The reaction mixture was concentrated under reduced pressure to remove THF, with H 2 O (500 mL) dilutes the residue and adjusts the pH to 3 with HCl (m=1), filters and concentrates under reduced pressure to afford compound 605 (260 g, crude) as a solid, which is used in the next step without further purification.
Preparation of Compound 606: trifluoroacetic acid (2.12 kg,18.6 mol) was added to CH at one time at 20℃under nitrogen 2 Cl 2 In a mixture of compound 605 (260 g,373 mmol) in (2.6L), and the mixture was stirred at 20℃for 5 hours. The reaction mixture was concentrated under reduced pressure and purified by HPLC (column Agela DuraShell C, 250 x 80mm x 10um; mobile phase: [ water (10 mm nh4hco 3) -MeOH]The method comprises the steps of carrying out a first treatment on the surface of the B%:5% -40%,20 min) to provide compound 606 (52.5 g) as a solid. (m+h) 642.80; IR 2107 (N) 3 A key).
Claims (33)
1. A method of functionalizing a nanoparticle, comprising:
contacting a nanoparticle with a first bifunctional precursor, wherein the first bifunctional precursor comprises a silane moiety and a diene moiety, wherein the nanoparticle comprises a surface reactive with silane (e.g., a silica surface), and wherein the contacting is performed under conditions suitable for a reaction between the silane moiety and the surface of the nanoparticle; and
Covalent bonds are formed between the silane moieties and the surface of the nanoparticle, thereby forming a nanoparticle functionalized with diene moieties.
2. The method of claim 1, further comprising:
contacting the nanoparticle functionalized with a diene moiety with a second bifunctional precursor, wherein the second bifunctional precursor comprises an alkyne moiety and a group that reacts with a diene moiety (e.g., a dienophile, e.g., maleimide), wherein the contacting is performed under conditions suitable for a reaction between the group that reacts with a diene moiety and the diene moiety; and
reacting the diene moiety of the nanoparticle with the second bifunctional precursor, thereby forming a nanoparticle functionalized with an alkyne moiety.
3. The method of claim 2, further comprising contacting the alkyne moiety with a compound comprising an alkyne reactive group (e.g., azide, diene, nitrone, or nitrile oxide) under conditions (e.g., click chemistry conditions) suitable for reaction of the alkyne moiety with the alkyne reactive group, thereby forming nanoparticles functionalized with the compound (e.g., a compound comprising a payload or a targeting ligand).
4. The method of any of the preceding claims, where the diene moiety is a cyclopentadiene moiety.
5. The method of any one of the preceding claims, wherein the first bifunctional precursor comprises a structure of formula (a):
wherein the method comprises the steps of
R 1 Is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen OR-OR A -wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl may be substituted or unsubstituted;
each R 2 Independently hydrogen, alkyl, halogen OR-OR A ;
R A Is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl;
n is an integer from 0 to 12; and
m is an integer from 0 to 5.
6. The method of claim 5, wherein
R 2 is-OR A ;
R A Is an alkyl group (e.g., ethyl);
n is an integer from 1 to 5 (e.g., 3); and
m is 0.
7. The method of any one of the preceding claims, wherein the first bifunctional precursor comprises a structure of formula (a-1):
8. the method of any one of claims 2-7, wherein the second dual function precursor comprises a structure of formula (B):
wherein:
x is a dienophile (e.g., a moiety comprising an electron-deficient alkene; or a cyclic dienophile, e.g., maleimide, quinone, or maleic anhydride);
Y is a divalent linker (e.g., substituted or unsubstituted heteroalkylene);
R 3 and R is 4 Each independently is alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, heteroaryl, halogen, -OR A 、-NR B R C 、-NO 2 -CN, wherein each alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl group may be substituted or unsubstituted;
R A 、R B 、R C each independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;
q and p are each independently integers from 0 to 4; and
v is an integer from 0 to 2.
9. The method of claim 8, wherein
X is maleimide;
q and p are each independently an integer 0; and
v is an integer of 1.
10. The method of claim 8 or 9, wherein the divalent linker is an alkylene or heteroalkylene group, wherein the alkylene or heteroalkylene group is unsubstituted or substituted (e.g., alkylene or heteroalkylene substituted with one or more oxo groups).
11. The method of any one of claims 8-10, wherein the divalent linker comprises a structure of formula (C):
wherein each R is B Independently is hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl;
x is an integer from 0 to 10; and
each of which isIndependently represents the point of attachment to a portion of the second bifunctional precursor (e.g., to the dienophile or to the nitrogen atom of the heterocycle).
12. The method of any one of claims 8-11, wherein the second dual-function precursor comprises a structure of formula (B-1):
where x is an integer from 0 to 10 (e.g., 4).
13. The method of claim 12, wherein reacting the diene moiety with the second bifunctional precursor provides a compound of formula (NP-1):
wherein x is an integer from 0 to 10 (e.g., 4), and wherein a silicon atom is part of the nanoparticle.
14. The method of any one of claims 3-13, wherein the compound comprising an alkyne reactive group comprises a payload moiety (e.g., a cytotoxic drug, e.g., isatecan) or a targeting ligand (e.g., a Folate Receptor (FR) targeting ligand, e.g., folic acid).
15. The method of any one of claims 3-14, wherein the compound comprising an alkyne reactive group comprises a structure of formula (D) or formula (E):
wherein:
t is a targeting ligand (e.g., FR targeting ligand, e.g., folic acid);
L is a cleavable linker moiety (e.g., a protease cleavable linker moiety);
p is a payload moiety (e.g., a cytotoxic drug, e.g., irinotecan); and
y is an integer from 0 to 20 (e.g., 3 or 9).
16. The method of claim 15, wherein the cleavable linker moiety comprises a structure of formula (F):
wherein the method comprises the steps of
Each instance of [ AA ] is a natural or unnatural amino acid residue;
z is an integer from 1 to 5;
w is an integer 2 or 3; and
each of which isIndependently represents a point of attachment to a portion of the alkyne-reactive group containing compound (e.g., to an azide group, or to a payload moiety).
17. The method of any one of claims 3-16, wherein the compound comprising an alkyne reactive group comprises a structure selected from the group consisting of:
wherein y is an integer from 0 to 20.
18. The method of any one of claims 3-17, wherein reacting the alkyne moiety with the compound comprising alkyne reactive groups provides a compound of formula (NP-2) or (NP-3):
wherein:
each instance of x is independently an integer from 0 to 10 (e.g., 4);
each instance of y is independently an integer from 0 to 20 (e.g., 3 or 9); and
Wherein the silicon atom in each of (NP-2) and (NP-3) is part of the nanoparticle.
19. The method of any one of the preceding claims, wherein the nanoparticle is a silica nanoparticle (e.g., a core-shell silica nanoparticle).
20. The method of any one of the preceding claims, wherein the nanoparticle (e.g., the surface of the nanoparticle) is coated with an organic polymer (e.g., polyethylene glycol).
21. The method of claim 20, wherein forming a covalent bond between the silane moiety of the first bifunctional precursor and the surface of the nanoparticle comprises inserting the first bifunctional precursor into interstitial spaces between organic polymer molecules (e.g., between PEG molecules) of the organic polymer.
22. The method of any of the preceding claims, wherein nanoparticles functionalized with multiple diene moieties are formed.
23. The method of claim 2, wherein nanoparticles functionalized with multiple alkyne moieties are formed.
24. The method of claim 23, further comprising:
(a) Reacting a first portion of the plurality of alkyne moieties with a first compound comprising alkyne reactive groups; and
(b) Reacting a second portion of the plurality of alkyne moieties with a second compound comprising alkyne reactive groups, thereby forming nanoparticles functionalized with the first compound and the second compound,
wherein the first compound and the second compound are chemically different.
25. The method according to claim 24, wherein:
(a) The first compound is a compound of formula (D) (e.g., a compound of formula (D-1)) and the second compound is a compound of formula (E) (e.g., a compound of formula (E-1)), or
(b) The first compound is a compound of formula (E) and the second compound is a compound of formula (D).
26. A method of functionalizing silica nanoparticles, comprising:
(i) Contacting a silica nanoparticle with a first bifunctional precursor, wherein the first bifunctional precursor comprises a silane moiety and a cyclopentadiene moiety, wherein the silica nanoparticle comprises a surface (e.g., a silica surface) that reacts with the silane moiety, and wherein the contacting is performed under conditions suitable for a reaction between the silane moiety and the silica nanoparticle surface, thereby forming a covalent bond between the silane moiety and the surface of the nanoparticle, and providing a nanoparticle functionalized with a cyclopentadiene moiety;
(ii) Contacting the nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor, wherein the second bifunctional precursor comprises an alkyne moiety (e.g., DBCO) and a dienophile (e.g., maleimide), wherein the contacting is performed under conditions suitable for a reaction between the dienophile and the cyclopentadiene moiety, thereby reacting the diene moiety of the nanoparticle with the second bifunctional precursor and providing the nanoparticle functionalized with an alkyne moiety; and
(iii) Contacting the alkyne moiety with a compound comprising an azide moiety under conditions suitable for reaction between the alkyne moiety and azide moiety (e.g., click chemistry conditions), thereby reacting the alkyne moiety of the nanoparticle with the azide moiety and providing a nanoparticle functionalized with the compound (e.g., a compound comprising a payload or a targeting ligand).
27. A method of functionalizing silica nanoparticles, comprising:
(i) Contacting the silica nanoparticle with a first bifunctional precursor comprising a structure of formula (a), thereby providing a nanoparticle functionalized with a cyclopentadiene moiety;
(ii) Contacting the nanoparticle functionalized with a cyclopentadiene moiety with a second bifunctional precursor comprising a structure of formula (B), thereby providing a nanoparticle functionalized with an alkyne moiety; and
(iii) Contacting the alkyne moiety with a compound of formula (D) or formula (E) or both, thereby providing a nanoparticle functionalized with a targeting ligand, a payload moiety, or both.
28. The method of claim 26 or 27, wherein the first bifunctional precursor comprises a structure of formula (a-1):
29. the method of any one of claims 26-28, wherein the second dual-function precursor comprises a structure of formula (B-1):
where x is an integer from 0 to 10 (e.g., 4).
30. The method of claim 27, wherein the compound of formula (D) comprises the structure of formula D-1:
where y is an integer from 0 to 10 (e.g., 3).
31. The method of claim 27, wherein the compound of formula (E) comprises the structure of formula E-1:
where y is an integer from 0 to 20 (e.g., 9).
32. The method of claim 29, wherein the method provides nanoparticles comprising a compound of formula (NP-2):
wherein x is 4 and y is 3, and a silicon atom is part of the nanoparticle.
33. The method of claim 31, wherein the method provides a compound of formula (NP-3):
wherein x is 4 and y is 9, and a silicon atom is part of the nanoparticle.
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