# Patent

## Title
Polycarbonates for delivery of drugs and methods of preparation thereof

## Abstract

A cyclic carbonate monomer has the formula (2):




wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional application No. 61/451,445 entitled “CHOLESTEROL-FUNCTIONAL POLYCARBONATES FOR DELIVERY OF ANTICANCER DRUGS” filed on Mar. 10, 2011, herein incorporated by reference in its entirety.


PARTIES TO A JOINT RESEARCH AGREEMENT
This invention was made under a joint research agreement between International Business Machines Corporation and the Agency For Science, Technology and Research.


BACKGROUND
The present invention relates to polycarbonates for delivery of drugs and methods of preparation thereof, and more specifically to block copolymers comprising polycarbonates bearing pendant steroidal groups for delivery of hydrophobic anticancer drugs.
One of the major problems in the development of anticancer drug formulations is the delivery of the drugs with adequately high bioavailability for therapeutic intention. As many anticancer agents are hydrophobic, clinical administration of these drugs typically requires dissolution using organic solvents. One such agent is paclitaxel, which is a widely used small molecular drug effective against an extensive range of solid tumors. However, its clinical applications have been mostly impeded by its extremely low aqueous solubility (0.3 micrograms/mL in water), which limits its administration to the use of a formulation comprising of 50:50 mixture of Cremophor EL (polyethoxylated castor oil):ethanol. Although this formulation is able to increase the solubility and bioavailability of paclitaxel (PTX), it can also lead to hypersensitivity reactions and other severe side effects in some cases. Despite premedication with corticosteroids to reduce the immune response, minor reactions such as rashes and flushing still occur in 41% to 44% of patients and potentially fatal reactions occur in 1.5% to 3% of patients.
Additional materials and methods are needed for administering hydrophobic drugs.
SUMMARY
Accordingly, a cyclic carbonate monomer is disclosed having the formula (2):




wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group.

Also disclosed is an amphiphilic block copolymer, comprising:
a hydrophilic poly(alkylene oxide) block; and
a biodegradable hydrophobic block comprising a first repeat unit of formula (3):




wherein i) t and t′ are integers independently having a value of 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, iv) S′ is a steroidal group.

Also disclosed is a loaded micelle comprising:
85.0 wt. % to 99.9 wt. % of the amphiphilic block copolymer comprising a) a hydrophilic poly(alkylene oxide) block and b) a biodegradable hydrophobic block comprising a carbonate repeat unit of formula (3):




wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group; and

15 wt. % to 0.1 wt. % of a biologically active cargo material bound by non-covalent interactions to the block copolymer, wherein weight percent (wt. %) is based on total dry weight of the loaded micelle.
Also disclosed is a method, comprising:
forming a solution comprising a water miscible organic solvent, a biologically active substance, and an amphiphilic block copolymer, the block copolymer comprising a) a hydrophilic poly(alkylene oxide) block and b) a biodegradable hydrophobic block comprising a carbonate repeat unit of formula (3):




wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group; and

dialyzing the solution against water using a dialysis membrane system, thereby forming a loaded micelle comprising the block copolymer bound by non-covalent interactions to the biologically active material.
Also disclosed is a method of treating a cell comprising contacting the cell with the above-described loaded micelle.
The above-described and other features and advantages of the present invention will be appreciated and understood by those skilled in the art from the following detailed description, drawings, and appended claims.



BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS
FIG. 1 is a graph showing the intensity ratio (I339/I334) versus logarithm of concentration (mg/L) of block copolymers (Examples 4, and 6 to 9), respectively, in deionized (DI) water. These data were used to determine critical micelle concentrations (CMC) values of Examples 4, and 6 to 9 of 2.1 mg/L, 2.1 mg/L, 1.5 mg/L, 1.5 mg/L and 2.1 mg/L, respectively (Table 8).
FIGS. 2A to 2F are transmission electron micrograph (TEM) images of aqueous micelles formed by the block copolymers of Examples 4 to 9, respectively. The upper image in FIG. 2F is at a magnification of 28000×. The lower image in FIG. 2F is at a magnification of 110000×.
FIG. 3 is a graph showing human liver carcinoma HepG2 cell viability (%) against block copolymer concentration (Examples 4, and 6 to 9). The block copolymers are non-cytotoxic. More than 90% cell viability was observed even at the highest polymer concentration (2377 mg/L).
FIGS. 4A to 4E are TEM images of paclitaxel loaded micelles (PTX-loaded micelles) of Examples 11 to 15.
FIG. 5A is a graph showing the cumulative release of PTX from the PTX-loaded micelles (Examples 11 to 15, Table 9) with time. Approximately 100% of the PTX was released within 24 hours from loaded micelles PTX-Polymer(11:0) (Example 12). PTX release was more sustained with the remaining loaded micelles (Example 11, and Examples 13 to 15), displaying 70% to 95% release by 144 hours.
FIG. 5B is a graph showing the cumulative release of the drug cyclosporin A (CYC) from the CYC-loaded micelles (Examples 16 to 18, Table 10) with time. CYC release was sustained from micellar formulation CYC-Polymer(11:30), Example 18, for about 5 days.
FIG. 5C is a graph showing the cumulative release of the drug spironolactone (SPL) from the SPL-loaded micelles (Examples 19 to 21, Table 11) with time. SPL release from micellar formulation SPL-Polymer(11:30), Example 21, was sustained for about 7 hours.
FIG. 6 is a bar chart comparing the viability of HepG2 cells after incubation with PTX-loaded micelles (Examples 11 to 15) and PTX alone for 48 hours at various PTX concentrations. The loaded micelle PTX-Polymer(8:8) (Example 13) killed HepG2 cells more efficiently than the free PTX at most PTX concentrations. A similar trend was observed with loaded micelle PTX-Polymer(11:0) (Example 12) at low PTX concentrations. The killing efficiency of loaded micelles PTX-Polymer(0:67) (Example 11), PTX-Polymer(11:30) (Example 14), and PTX-Polymer(18:55) (Example 15) against HepG2 cells was greater than free PTX at all concentrations tested.



DETAILED DESCRIPTION
The invention is based on cyclic carbonate monomers comprising pendant steroidal groups, referred to herein as steroidal monomers, which undergo organocatalyzed ring-opening polymerization (ROP) to form amphiphilic biodegradable and/or biocompatible copolymers functionalized with a side chain steroidal group. The polymer chain formed by ring opening polymerization is preferably a copolymer of a steroidal monomer and one or more diluent cyclic carbonate monomers (e.g., trimethylene carbonate (TMC)) and/or cyclic ester monomers (i.e., lactones and/or lactides). The ring opening polymerization is preferably initiated using a hydrophilic polymeric initiator having one or more nucleophilic initiator groups selected from the group consisting of alcohols, amines, thiols and combinations thereof, which results in an amphiphilic block copolymer. The amphiphilic block copolymers have utility as injectable delivery systems for hydrophobic drugs and/or genes.
The amphiphilic block copolymers can have two or more blocks. In an embodiment, the polymeric initiator is a mono-endcapped poly(alkylene glycol), such as mono-methyl poly(ethylene glycol) (mPEG-OH)). The amphiphilic block copolymer derived from mPEG-OH comprises a poly(alkylene oxide) block and a polycarbonate and/or polyestercarbonate block bearing a side chain steroidal group. For a given mono-endcapped poly(alkylene glycol) initiator, the amphiphilic properties of the block copolymers can be controlled by adjusting the amount and structure of the various repeat units in the ring opened polymer chain, that is, by adjusting the amount and structure of the cyclic carbonate monomer bearing a steroidal group and/or the amount and structure of a diluent cyclic carbonate and/or cyclic ester monomer(s) used in the ring opening polymerization. The diluent monomer does not comprise a steroidal group.
The amphiphilic block copolymers have low critical micelle concentrations (CMCs) in water. The CMC can have a value of 1.0 mg/L to 50 mg/L, 1.0 mg/L to 10 mg/L, and more particularly 1.0 mg/L to 2.5 mg/L.
In aqueous media, the amphiphilic block copolymers are capable of forming by noncovalent interactions reversible nanoparticulate complexes with hydrophobic biologically active substances (e.g., drugs and/or genes). For example, the amphiphilic block copolymers can encapsulate the rigid hydrophobic anticancer drug paclitaxel (PTX) in the form of a water dispersible nano-sized particle (i.e., loaded micelle). Paclitaxel has the structure:




The loaded micelle forms without sonication or homogenization and has a narrow particle size distribution. The loaded micelle comprises PTX in an amount more than 0 wt. %, and more particularly in an amount of 0.1 wt. % to 15 wt. % based on total dry weight of the loaded micelle. Other hydrophobic drugs, which can be encapsulated by the amphiphilic block copolymers include cyclosporin A (CYC, an immunosuppressive agent used after organ transplantation) and spironolactone (SPL, a drug used for hair growth). The drugs can be used singularly or in combination. CYC and SPL have the structures (stereochemistry shown):





In the following description, the term “cyclic carbonyl monomer” includes cyclic carbonate monomers, cyclic ester monomers, and combinations thereof that can be polymerized in a ring opening polymerization.
Herein, a cyclic carbonate monomer having a pendant steroidal group is referred to as a “steroidal monomer.” A second cyclic carbonyl monomer, referred to as a “diluent monomer,” does not contain a steroidal group. The diluent monomer can be a cyclic carbonate monomer, a cyclic ester (i.e., lactone, lactide) monomer, or a combination thereof.
The amphiphilic block copolymers preferably comprise a hydrophilic poly(alkylene oxide) block and a hydrophobic block comprising i) a first repeat unit comprising a backbone carbonate group and a side chain comprising a steroidal group and ii) a second repeat unit comprising a backbone carbonate group. The second repeat unit does not comprise a steroidal group. The second repeat units can be substituted or unsubstituted. Preferably, the amphiphilic block copolymers are also biodegradable and/or biocompatible.
The term “biodegradable” is defined by the American Society for Testing and Materials as degradation caused by biological activity, especially by enzymatic action, leading to a significant change in the chemical structure of the material. For purposes herein, a material is “biodegradable” if it undergoes 60% biodegradation within 180 days in accordance with ASTM D6400. Herein, a material is “enzymatically biodegradable” if the material can be degraded (e.g., depolymerized) by a reaction catalyzed by an enzyme.
A “biocompatible” material is defined herein as a material capable of performing with an appropriate host response in a specific application.
The steroidal monomer and the diluent monomer can be stereospecific or non-stereospecific. A stereospecific monomer or a stereospecific repeat unit i) has a non-superposable mirror image and ii) comprises one or more asymmetric tetravalent carbons (i.e., tetrahedral sp3 carbons). Each asymmetric tetravalent carbon is assigned an R or S symmetry based on Cahn-Ingold-Prelog (CIP) symmetry rules. For example, if a stereospecific repeat unit has one asymmetric tetravalent carbon, then the stereospecific repeat unit can be present substantially as the R stereoisomer or substantially as the S stereoisomer, meaning the stereoisomer can be present in a stereoisomeric purity of 90% to 100%, 94% or more, or more particularly 98% to 100%. In another example, if the stereospecific repeat unit has two asymmetric tetravalent carbons, the stereospecific repeat unit can be present substantially as the R,R stereoisomer, substantially as the R,S stereoisomer, substantially as the S,S stereoisomer, or substantially as the S,R stereoisomer.
A stereospecific cyclic carbonyl monomer i) has a non-superposable mirror image and ii) comprises one or more asymmetric tetravalent carbons. The stereospecific cyclic carbonyl monomer has a stereoisomeric purity of 90% or more, and more particularly 98% or more. The asymmetric tetravalent carbons of the stereospecific cyclic carbonyl monomer can be in the steroidal group or in a ring carbon that becomes a polymer backbone carbon in a ring opening polymerization.
“Restricted metals” herein include ionic and nonionic forms of beryllium, magnesium, calcium, strontium, barium, radium, aluminum, gallium, indium, thallium, germanium, tin, lead, arsenic, antimony, bismuth, tellurium, polonium, and metals of Groups 3 to 12 of the Periodic Table. Metals of Groups 3 to 12 of the Periodic Table include scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, zinc, yttrium, zirconium, niobium, molybdenum, technetium, ruthenium, rhodium, palladium, silver, cadmium, lanthanum, cerium, praseodymium, neodymium, promethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, hafnium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, actinium, thorium, protactinium, uranium, neptunium, plutonium, americium, curium, berkelium, californium, einsteinium, fermium, mendelevium, nobelium, lawrencium, rutherfordium, dubnium, seaborgium, bohrium, hassium, meitnerium, darmstadtium, roentgenium, and copernicium. Each one of the foregoing restricted metals can have a concentration in the block copolymer of 0 parts to 100 ppm (parts per million), 0 parts to 100 ppb (parts per billion), or 0 parts to 100 ppt (parts per trillion). Preferably, each one of the foregoing restricted metals has a concentration of 0 parts in the amphiphilic block copolymer (i.e., the concentration is below detection limits). In an embodiment, the chemical formulas of the components used in the ring opening polymerization, including the steroidal monomer, the diluent monomer, the mono-nucleophilic polymeric initiator, the catalyst for the ring opening polymerization, the solvent, and any base accelerator, contain none of the above restricted metals. The biologically active cargo material can comprise a restricted metal.
No restriction is placed on the concentration of boron, silicon, or any individual alkali metal in the disclosed block copolymer compositions.
The term “carrier” as used herein refers to the amphiphilic block copolymer. The carrier can be biologically active when used alone (e.g., as a result of enzymatic release of the covalently bound steroidal group, where the released steroid performs a therapeutic function). A loaded micelle can therefore serve multiple biological functions, including providing enhanced biological activity of either the released steroid and/or the released biologically active substance (i.e., cargo material). As one example, the loaded micelle can enter the blood stream by injection and release the biologically active substance over a desired period. Alternatively or additionally, the loaded micelle can enter a tissue or a cell (e.g., by endocytosis), and at a desired stage release the biologically active substance within the tissue or the cell.
The biologically active substance can be any suitable biologically active substance that complexes with the amphiphilic block copolymer in water by non-covalent interactions to form a loaded micelle. Biologically active substances include cells, biomolecules (e.g., DNA, genes, peptides, proteins, enzymes, lipids, phospholipids, and nucleotides), natural or synthetic organic compounds (e.g., drugs, dyes, synthetic polymers, oligomers, and amino acids), inorganic materials (e.g., metals and metal oxides), radioactive variants of the foregoing, and combinations of the foregoing. In an embodiment, the biologically active substance is a drug and/or a gene.
“Biologically active” means the referenced material can alter the chemical structure and/or activity of a cell in a desirable manner, or can selectively alter the chemical structure and/or activity of a cell type relative to another cell type in a desirable manner. As an example, one desirable change in a chemical structure can be the incorporation of a gene into the DNA of the cell. A desirable change in activity can be the expression of the transfected gene. Another change in cell activity can be the induced production of a desired hormone or enzyme. Alternatively, a desirable change in activity can be the selective death of one cell type over another cell type. No limitation is placed on the relative change in cellular activity caused by the biologically active substance, providing the change is desirable and useful. Moreover, no limitation is placed on the cargo, providing the cargo induces a useful cellular response when released from the loaded micelle.
The steroidal monomer is a cyclic carbonate monomer comprising a pendant steroidal group. The steroidal group can be a chemical moiety derived from a naturally occurring human steroid, non-human steroid, and/or synthetic steroid compound. Herein, a steroidal group comprises a tetracyclic ring structure according to formula (1):




wherein the 17 carbons of the ring system are numbered as shown. Each ring of the tetracyclic ring structure can independently comprise one or more double bonds. The steroidal group can comprise one or more monovalent substituent groups independently comprising 0 to 30 carbons attached to one or more of the numbered ring positions of the tetracyclic ring structure.

The steroidal monomer comprises a cyclic carbonate ring which is joined to a steroidal group by a divalent linking group L′. The steroidal monomer has the formula (2):




wherein t and t′ are independent integers having a value of 0 to 6 wherein t′ and t cannot both be zero, and each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms. Ring carbons and oxygens are numbered as shown in formula (2). L′ is a divalent linking group comprising one or more carbons. S′ is a steroidal group. The bond joining L′-S′ bond is preferably hydrolytically cleavable and/or enzymatically cleavable, meaning the bond can be directly or indirectly cleaved as a result of enzymatic activity. Each Q1 group can independently be branched or non-branched. Each Q1 group can also independently comprise one or more additional functional groups selected from the group consisting of ketones, aldehydes, alkenes, alkynes, cycloaliphatic rings comprising 3 to 10 carbons, heterocylic rings comprising 2 to 10 carbons, ethers, amides, esters, and combinations of the foregoing functional groups. A heterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two or more Q1 groups can together form a ring. The steroidal monomer can be stereospecific or non-stereospecific. In an embodiment, t and t′ are each 1, each Q1 at carbon 4 is hydrogen, each Q1 at carbon 6 is hydrogen, and Q1 at carbon 5 is selected from the group consisting of hydrogen, methyl, and ethyl. In another embodiment, S′ is a cholesteryl group.

L′ can comprise one or more functional groups selected from the group consisting of ketone, ester, amide, imide, thioester, carbamate, thiocarbamate (i.e., —S—C═O(N(R)—), urea, anhydride, and combinations thereof. In an embodiment, L′ comprises a carbonyl group linked to S′. In another embodiment, L′ comprises an amidocarbonyl group:




wherein R′ is hydrogen or a monovalent radical comprising 1 to 5 carbons, and the carbonyl group of the amidocarbonyl group is linked to S′.

Exemplary steroidal groups S′ include but are not limited to stereospecific structures such as:




from cholesterol and referred to herein as a cholesteryl group,





from testosterone,





from equilin,





from epiandrosterone,





from dihydrotestosterone,





from nandrolone,





from dihydroprogesterone,





from pregnenolone,





from equilenin,





from dehydroepiandrosterone,





from corticosterone acetate,





from deoxycorticosterone, and





from estrone.

In the above steroidal groups, the starred bond represents the attachment point to L′. The R,S stereochemistry of each asymmetric tetravalent carbon is indicated in the above steroidal groups. Other steroidal groups include various stereoisomers of the foregoing steroidal groups. The steroidal groups can be used in combination, meaning two or more steroidal monomers differing in chemical structure and/or in stereospecificity can be used to form the polycarbonate block of the amphiphilic block copolymer.

Ring opening polymerization of the steroidal monomer of formula (2) produces a first repeat unit having the formula (3):




wherein L′, t, t′, Q1, and S′ are defined as above. Atom numbers of the backbone carbons and oxygens are indicated in formula (3). The first repeat unit comprises a backbone carbonate group. The starred bonds in formula (3) represent attachment points to other subunits or to a terminal functional group of the ROP polymer chain.

More specific steroidal monomers include cyclic carbonate compounds of formula (4):




wherein t, t′, Q1, and S′ are defined as above. L′′ can be a single bond (i.e., S′ is linked to the ester oxygen of formula (4) by a single bond). Alternatively, L′′ can be a divalent linking group comprising 1 to 30 carbons. The L′′-S′ bond can be a hydrolytically and/or enzymatically cleavable bond.

Ring opening polymerization of the steroidal monomer of formula (4) produces a first repeat unit having the formula (5):




wherein L′′, t, t′, Q1, and S′ are defined as above, and backbone carbons and oxygens are numbered as indicated. The first repeat unit comprises a backbone carbonate group.

More specific steroidal monomers of formula (2) include cholesteryl monomers of formula (6):




wherein L′, t, t′ and Q1 are defined as in formula (2). The R,S stereochemistry of each asymmetric tetravalent carbon center of the cholesteryl moiety is indicated in formula (6). In an embodiment, the L′-O bond in formula (6) is hydrolytically and/or enzymatically cleavable.

The first repeat unit formed by a ring opening polymerization of the cholesteryl monomer of formula (6) has the formula (7):




wherein L′, t, t′, and Q1 are defined as in formula (2), and backbone carbons and oxygens are numbered as shown. The R,S stereochemistry of each asymmetric tetravalent carbon center of the cholesteryl moiety is indicated in formula (7). The first repeat unit of formula (7) comprises a backbone carbonate group.

More specific steroidal monomers of formula (4) include cholesteryl monomers of formula (8):




with R,S stereochemistry shown, wherein L′′ is defined as above under formula (4), and t, t′, and Q1 are defined as above under formula (2).

The first repeat unit formed by ring opening polymerization of the cholesteryl monomer of formula (8) has the formula (9):




with R,S stereochemistry shown, wherein L′′, t, t′, and Q1 are defined as above, and backbone carbons and oxygens are numbered as shown. The first repeat unit of formula (9) comprises a backbone carbonate group.

In an embodiment, L′ has a structure according to formula (10):




wherein L′′′ is a divalent linking group comprising 1 to 20 carbons. L′′′ can comprise one or more heteroatoms selected from the group consisting of oxygen, nitrogen, sulfur, and combinations thereof. In an embodiment L′′′ has the formula *—O—CH2(CH2)xCH2N(R)—*, wherein x is an integer of 1 to 10 and R′ is an monovalent alkyl group having 1 to 5 carbons:

The diluent monomer, which forms a second repeat unit of the ROP polymer chain, can be selected from cyclic carbonate compounds of the formula (11):




wherein u and u′ are independent integers having a value of 0 to 6 wherein u and u′ cannot both be zero, and each Q2 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms. Ring carbons and oxygens are numbered as shown in formula (11). No Q2 group comprises a steroidal group. Each Q2 group can independently be branched or non-branched. Each Q2 group can independently be stereospecific or non-stereospecific. Each Q2 group can independently comprise one or more additional functional groups selected from the group consisting of ketones, aldehydes, alkenes, alkynes, ethers, amides, esters, cycloaliphatic rings comprising 3 to 10 carbons, heterocylic rings comprising 2 to 10 carbons, and combinations of the foregoing functional groups. A heterocyclic ring can comprise oxygen, sulfur and/or nitrogen. Two or more Q2 groups can together form a ring. In an embodiment, u′ and u are each 1, and each Q2 is hydrogen (i.e., trimethylene carbonate).

The second repeat unit formed by ring opening polymerization of the diluent monomer of formula (11) has the formula (12):




wherein u, u′, and Q2 are defined as above under formula (11), and backbone carbons and oxygens are numbered as shown. The second repeat unit comprises a backbone carbonate group.

More specific diluent monomers of formula (11) have the formula (13):




wherein Q2 is defined as above under formula (11), R2 is a monovalent radical independently selected from the group consisting of alkyl groups comprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons, and R2 does not comprise a steroidal group.

The second repeat unit formed by ring opening polymerization of the diluent monomer of formula (13) has the formula (14):




wherein Q2 and R2 are defined as above, and backbone carbons and oxygens are numbered as shown. The second repeat unit comprises a backbone carbonate group.

The diluent monomer can be selected from cyclic ester monomers (e.g., lactones). Exemplary cyclic ester monomers include compounds of the formula (15):




wherein v is an integer of 1 to 8, each Q3 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms. No Q3 group comprises a steroidal group. The cyclic ester ring can optionally comprise a carbon-carbon double bond; that is, optionally, a





group of formula (15) can independently represent a





group. The cyclic ester ring can also comprise a heteroatom such as oxygen, nitrogen, sulfur, or a combination thereof; that is, optionally a





group of formula (15) can independently represent a *—O—*, *—S—*, *—N(H)—*, or an *—N(R1)—* group, wherein R1 is a monovalent radical independently selected from the group consisting of alkyl groups comprising 1 to 30 carbons and aryl groups comprising 6 to 30 carbons. Cyclic ester monomers of formula (15) can be stereospecific or non-stereospecific.

The second repeat unit formed by ring opening polymerization of the diluent monomer of formula (15) has the formula (16):




wherein Q3 and v are defined as above under formula (15). The second repeat unit comprises a backbone ester group.

A ring opened polymer chain that comprises a first repeat unit comprising a backbone carbonate group and a second repeat unit comprising a backbone carbonate group is a polycarbonate. A ring opened polymer chain that comprises a first repeat unit comprising a backbone carbonate group and second repeat unit comprising a backbone ester group is referred to herein as a polyestercarbonate.
The diluent monomer can be selected from a dioxane dicarbonyl monomers of the formula (17):




wherein w and w′ are independent integers having a value of 1 to 3, and each Q4 and each Q5 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms. No Q4 group and no Q5 group comprises a steroidal group. Compounds of formula (17) can be stereospecific or non-stereospecific. In an embodiment, w and w′ are each 1, each Q4 is hydrogen, and each Q5 is an alkyl group comprising 1 to 6 carbons. In another embodiment, the diluent monomer is D-lactide or L-lactide.

The second repeat unit formed by ring opening polymerization of the diluent monomer of formula (17) has the formula (18):




wherein Q4, Q5, w, and w′ are defined as above under formula (17), and backbone carbons and oxygens are numbered as shown. This second repeat unit has two backbone ester groups.

Non-limiting examples of diluent monomers of formulas (11) and (13) include the cyclic carbonate monomers of Table 1.







TABLE 1













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















Non-limiting examples of diluent monomers having a cyclic ester structure of formula (15) include the compounds of Table 2, and stereospecific versions thereof where feasible.







TABLE 2













 








 








 








 








 








 








 








 








 








 















Examples of diluent monomers of formula (17) include the compounds of Table 3.







TABLE 3













 








 















The above monomers can be purified by recrystallization from a solvent such as ethyl acetate or by other known methods of purification, with particular attention being paid to removing as much water as possible from the monomer. The monomer moisture content can have a value of 1 to 10,000 ppm, 1 to 1,000 ppm, 1 to 500 ppm, and most specifically 1 to 100 ppm, by weight of the monomer.
ROP Initiators.
In general, initiators for ring opening polymerizations include nucleophilic groups such as alcohols, primary amines, secondary amines, and thiols. Initiators can comprise one or more nucleophilic initiator groups, as appropriate. The initiator can be a monomer, oligomer, or a polymeric initiator based on the intended structure of the ring opened polymer. The initiator can include other functional groups, including protected nucleophilic groups that include protected thiols, protected amines, and protected alcohols. Exemplary monomeric mono-nucleophilic initiators include mono-alcohols, such as methanol, ethanol, propanol, butanol, pentanol, amyl alcohol, capryl alcohol, nonyl alcohol, decyl alcohol, undecyl alcohol, lauryl alcohol, tridecyl alcohol, myristyl alcohol, pentadecyl alcohol, cetyl alcohol, heptadecyl alcohol, stearyl alcohol, nonadecyl alcohol and other aliphatic saturated alcohols, cyclopentanol, cyclohexanol, cycloheptanol, cyclooctanol and other aliphatic cyclic alcohols; phenol, substituted phenols, benzyl alcohol, substituted benzyl alcohols, and the like. Exemplary polymeric mono-nucleophilic initiators include mono-endcapped poly(ethylene glycols), and mono-endcapped poly(propylene glycols). Exemplary monomeric and oligomeric dinucleophilic initiators include benzenedimethanol, hydroquinone, resorcinol, propylene glycol, ethylene glycol, diethylene glycol, and triethylene glycol. Other dinucleophilic initiators include monomeric diols such as 1,3-propanediol, 1,4-butanediol, 1,5-pentanediol, 1,6-hexanediol, and the like. An even more specific dinucleophilic initiator is BnMPA, a precursor used in the preparation of cyclic carbonate monomers:




Preferably, the initiator for the ring opening polymerization is a hydrophilic mono-nucleophilic polyether initiator. The polyether initiator preferably comprises a mono-endcapped poly(alkylene glycol) of the formula (19):

E′-[CH2(CHR1)xCHR1O]n-1—H  (19),

wherein E′ represents a non-nucleophilic derivative of a terminal hydroxy alkylene subunit (i.e., E′ is a non-nucleophilic derivative of HOCH2(CHR1)xCHR1O—*), x is 0 or 1, n is an integer from 2 to 10000, and each R1 is hydrogen or methyl. Thus, the ether repeat unit can comprise 2 or 3 backbone carbons between each backbone oxygen in the hydrophilic block of the block copolymer. Non-limiting examples of hydrophilic polyether initiators include mono-methyl endcapped poly(ethylene glycol) (PEG), having the structure MeO—CH2CH2O—[CH2CH2O]n-1—H, and mono-methyl endcapped poly(propylene glycol) (PPG) having the structure MeO—CH2C(H)(CH3)O—[CH2C(H)(CH3)O]n-1—H, and polyether initiators comprising a mixture of ethyleneoxy and propyleneoxy repeat units. E′ does not comprise an active initiator group for a ring opening polymerization. In an embodiment, E′ comprises a biologically active moiety.

The polymeric initiator can comprise a nucleophilic chain end group independently selected from the group consisting alcohols, primary amines, secondary amines, and thiols, such as mono-endcapped PEG-diamine represented by the structure




wherein E′′ represents a derivative of a terminal aminoethylene group, and monoendcapped PEG-dithiol, represented by the formula





wherein E′′′ comprises a derivative of a terminal thioethylene group.

The number average molecular weight (Mn) of the mono-nucleophilic polyether initiator can have a value of 100 to 100,000, more preferably 100 to 10000, and even more preferably 100 to 5000.
Ring Opening Polymerizations (ROP).
The following description of methods, conditions and materials for ring opening polymerizations is applicable to the preparation of the block copolymer.
The ring-opening polymerization can be performed at a temperature that is about ambient temperature or higher, more specifically 15° C. to 200° C., and even more specifically 20° C. to 80° C. When the reaction is conducted in bulk, the polymerization is performed at a temperature of 50° C. or higher, and more particularly 100° C. to 200° C. Reaction times vary with solvent, temperature, agitation rate, pressure, and equipment, but in general the polymerizations are complete within 1 to 100 hours.
The ROP reaction can be performed with or without the use of a solvent. Optional solvents include dichloromethane, chloroform, benzene, toluene, xylene, chlorobenzene, dichlorobenzene, benzotrifluoride, petroleum ether, acetonitrile, pentane, hexane, heptane, 2,2,4-trimethylpentane, cyclohexane, diethyl ether, t-butyl methyl ether, diisopropyl ether, dioxane, tetrahydrofuran, or a combination comprising one of the foregoing solvents. When a solvent is present, a suitable monomer concentration is about 0.1 to 5 moles per liter, and more particularly about 0.2 to 4 moles per liter.
Whether performed in solution or in bulk, the ROP polymerizations are conducted under an inert (i.e., dry) atmosphere, such as nitrogen or argon, and at a pressure of 100 MPa to 500 MPa (1 atm to 5 atm), more typically at a pressure of 100 MPa to 200 MPa (1 atm to 2 atm). At the completion of the reaction, the solvent can be removed using reduced pressure.
Less preferred catalysts for the ROP polymerization include metal oxides such as tetramethoxy zirconium, tetra-iso-propoxy zirconium, tetra-iso-butoxy zirconium, tetra-n-butoxy zirconium, tetra-t-butoxy zirconium, triethoxy aluminum, tri-n-propoxy aluminum, tri-iso-propoxy aluminum, tri-n-butoxy aluminum, tri-iso-butoxy aluminum, tri-sec-butoxy aluminum, mono-sec-butoxy-di-iso-propoxy aluminum, ethyl acetoacetate aluminum diisopropylate, aluminum tris(ethyl acetoacetate), tetraethoxy titanium, tetra-iso-propoxy titanium, tetra-n-propoxy titanium, tetra-n-butoxy titanium, tetra-sec-butoxy titanium, tetra-t-butoxy titanium, tri-iso-propoxy gallium, tri-iso-propoxy antimony, tri-iso-butoxy antimony, trimethoxy boron, triethoxy boron, tri-iso-propoxy boron, tri-n-propoxy boron, tri-iso-butoxy boron, tri-n-butoxy boron, tri-sec-butoxy boron, tri-t-butoxy boron, tri-iso-propoxy gallium, tetramethoxy germanium, tetraethoxy germanium, tetra-iso-propoxy germanium, tetra-n-propoxy germanium, tetra-iso-butoxy germanium, tetra-n-butoxy germanium, tetra-sec-butoxy germanium and tetra-t-butoxy germanium; halogenated compound such as antimony pentachloride, zinc chloride, lithium bromide, tin(IV) chloride, cadmium chloride and boron trifluoride diethyl ether; alkyl aluminum such as trimethyl aluminum, triethyl aluminum, diethyl aluminum chloride, ethyl aluminum dichloride and tri-iso-butyl aluminum; alkyl zinc such as dimethyl zinc, diethyl zinc and diisopropyl zinc; heteropolyacids such as phosphotungstic acid, phosphomolybdic acid, silicotungstic acid and alkali metal salt thereof; zirconium compounds such as zirconium acid chloride, zirconium octanoate, zirconium stearate, and zirconium nitrate.
The catalyst is preferably an organocatalyst whose chemical formula contains none of the restricted metals described further above. Examples of organocatalysts for ring opening polymerizations include tertiary amines such as triallylamine, triethylamine, tri-n-octylamine and benzyldimethylamine 4-dimethylaminopyridine, phosphines, N-heterocyclic carbenes (NHC), bifunctional aminothioureas, phosphazenes, amidines, and guanidines.
A more specific organocatalyst is N-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU):




Other ROP organocatalysts comprise at least one 1,1,1,3,3,3-hexafluoropropan-2-ol-2-yl (HFP) group. Singly-donating hydrogen bond catalysts have the formula (20):

R2—C(CF3)2OH  (20),

wherein R2 represents a hydrogen or a monovalent radical having 1 to 20 carbons, for example an alkyl group, substituted alkyl group, cycloalkyl group, substituted cycloalkyl group, heterocycloalkyl group, substituted heterocycloalklyl group, aryl group, substituted aryl group, or a combination thereof. Exemplary singly-donating hydrogen bonding catalysts are listed in Table 4.








TABLE 4













 















Doubly-donating hydrogen bonding catalysts have two HFP groups, represented by the formula (21):




wherein R3 is a divalent radical bridging group comprising 1 to 20 carbons, such as an alkylene group, a substituted alkylene group, a cycloalkylene group, substituted cycloalkylene group, a heterocycloalkylene group, substituted heterocycloalkylene group, an arylene group, a substituted arylene group, and a combination thereof. Representative double hydrogen bonding catalysts of formula (21) include those listed in Table 5. In a specific embodiment, R2 is an arylene or substituted arylene group, and the HFP groups occupy positions meta to each other on the aromatic ring.








TABLE 5













 








 








 















In one embodiment, the catalyst is selected from the group consisting of 4-HFA-St, 4-HFA-Tol, HFTB, NFTB, HPIP, 3,5-HFA-MA, 3,5-HFA-St, 1,3-HFAB, 1,4-HFAB, and combinations thereof.
Also contemplated are catalysts comprising HFP-containing groups bound to a support. In one embodiment, the support comprises a polymer, a crosslinked polymer bead, an inorganic particle, or a metallic particle. HFP-containing polymers can be formed by known methods including direct polymerization of an HFP-containing monomer (for example, the methacrylate monomer 3,5-HFA-MA or the styryl monomer 3,5-HFA-St). Functional groups in HFP-containing monomers that can undergo direct polymerization (or polymerization with a comonomer) include acrylate, methacrylate, alpha, alpha, alpha-trifluoromethacrylate, alpha-halomethacrylate, acrylamido, methacrylamido, norbornene, vinyl, vinyl ether, and other groups known in the art. Examples of linking groups include C1-C12 alkyl, a C1-C12 heteroalkyl, ether group, thioether group, amino group, ester group, amide group, or a combination thereof. Also contemplated are catalysts comprising charged HFP-containing groups bound by ionic association to oppositely charged sites on a polymer or a support surface.
The ROP reaction mixture comprises at least one organocatalyst and, when appropriate, several organocatalysts together. The ROP catalyst is added in a proportion of 1/20 to 1/40,000 moles relative to the cyclic carbonyl monomers, and preferably in a proportion of 1/1,000 to 1/20,000 moles relative to the cyclic carbonyl monomers.
ROP Accelerators.
The ROP polymerization can be conducted in the presence of an optional accelerator, in particular a nitrogen base. Exemplary nitrogen base accelerators are listed below and include pyridine (Py), N,N-dimethylaminocyclohexane (Me2NCy), 4-N,N-dimethylaminopyridine (DMAP), trans 1,2-bis(dimethylamino)cyclohexane (TMCHD), 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,5,7-triazabicyclo[4.4.0]dec-5-ene (TBD), 7-methyl-1,5,7-triazabicyclo[4.4.0]dec-5-ene (MTBD), (−)-sparteine, (Sp) 1,3-bis(2-propyl)-4,5-dimethylimidazol-2-ylidene (Im-1), 1,3-bis(2,4,6-trimethylphenyl)imidazol-2-ylidene (Im-2), 1,3-bis(2,6-di-1-propylphenyl(imidazol-2-ylidene (Im-3), 1,3-bis(1-adamantyl)imidazol-2-ylidene (Im-4), 1,3-di-1-propylimidazol-2-ylidene (Im-5), 1,3-di-t-butylimidazol-2-ylidene (Im-6), 1,3-bis(2,4,6-trimethylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-7), 1,3-bis(2,6-di-1-propylphenyl)-4,5-dihydroimidazol-2-ylidene, 1,3-bis(2,6-di-i-propylphenyl)-4,5-dihydroimidazol-2-ylidene (Im-8) or a combination thereof, shown in Table 6.







TABLE 6













 








 








 








 








 








 








 








 








 








 








 








 








 








 








 















In an embodiment, the accelerator has two or three nitrogens, each capable of participating as a Lewis base, as for example in the structure (−)-sparteine. Stronger bases generally improve the polymerization rate.
The catalyst and the accelerator can be the same material. For example, some ring opening polymerizations can be conducted using 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) alone, with no another catalyst or accelerator present.
The catalyst is preferably present in an amount of about 0.2 to 20 mol %, 0.5 to 10 mol %, 1 to 5 mol %, or 1 to 2.5 mol %, based on total moles of cyclic carbonyl monomer.
The nitrogen base accelerator, when used, is preferably present in an amount of 0.1 to 5.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, or 0.2 to 0.5 mol %, based on total moles of cyclic carbonyl monomer. As stated above, in some instances the catalyst and the nitrogen base accelerator can be the same compound, depending on the particular cyclic carbonyl monomer.
The amount of initiator is calculated based on the equivalent molecular weight per nucleophilic initiator group. The initiator groups are preferably present in an amount of 0.001 to 10.0 mol %, 0.1 to 2.5 mol %, 0.1 to 1.0 mol %, and 0.2 to 0.5 mol %, based on total moles of cyclic carbonyl monomer. For example, if the molecular weight of the initiator is 100 g/mole and the initiator has 2 hydroxyl groups, the equivalent molecular weight per hydroxyl group is 50 g/mole. If the polymerization calls for 5 mol % hydroxyl groups per mole of cyclic carbonyl monomer, the amount of initiator is 0.05×50=2.5 g per mole of cyclic carbonyl monomer.
In a specific embodiment, the catalyst is present in an amount of about 0.2 to 20 mol %, the nitrogen base accelerator is present in an amount of 0.1 to 5.0 mol %, and the nucleophilic initiator groups of the initiator are present in an amount of 0.1 to 5.0 mol % based on the equivalent molecular weight per nucleophilic initiator group of the initiator.
The catalysts can be removed by selective precipitation or in the case of the solid supported catalysts, simply by filtration. The block copolymer can comprise residual catalyst in an amount greater than 0 wt. % (weight percent), based on total weight of the block copolymer and the residual catalyst. The amount of residual catalyst can also be less than 20 wt. %, less than 15 wt. %, less than 10 wt. %, less than 5 wt. %, less than 1 wt. %, or most specifically less than 0.5 wt. % based on the total weight of the block copolymer and the residual catalyst.
Average Molecular Weight.
The block copolymer preferably has a number average molecular weight (Mn) as determined by size exclusion chromatography of at least 1500 g/mol, more specifically 1500 g/mol to 1,000,000 g/mol, 4000 g/mol to 150000 g/mol, or 4000 g/mol to 50000 g/mol. In an embodiment, the block copolymer has a number average molecular weight Mn of 10,000 to 20,000 g/mole. The block copolymer preferably has a narrow polydispersity index (PDI) of 1.01 to 2.0, more particularly 1.01 to 1.30, and even more particularly 1.01 to 1.25.
Endcap Agents.
The ring opened polymer can further be treated with an endcap agent to prevent further chain growth and stabilize the reactive end groups against unwanted side reactions such as chain scission. Endcap agents include, for example, materials for converting terminal hydroxyl groups to esters, such as carboxylic acid anhydrides, carboxylic acid chlorides, or reactive esters (e.g., p-nitrophenyl esters). In an embodiment, the endcap agent is acetic anhydride, which converts reactive hydroxy end groups to acetate ester groups. The endcap agent can also comprise a biologically active moiety, which becomes bound to the terminal end group of the ring opened polymer chain.
Cytotoxicity of the Block Copolymer.
The amphiphilic block copolymers alone are generally non-cytotoxic. For example, cell viability of HepG2, a human liver carcinoma cell line, is in a range of 95% or more at block copolymer concentrations of 470 mg/L, or higher.
Loaded Micelles.
In water optionally containing organic solvent, the block copolymer self-assembles to form a nanoparticulate micelle solution. When a biologically active cargo material is also present, the block copolymer and cargo material form a loaded micelle bound by non-covalent interactions.
A method comprises i) forming a solution of an amphiphilic block copolymer (i.e., carrier) and a biologically active material (i.e., cargo) in a water miscible organic solvent, ii) dialyzing the solution against deionized water using a dialysis membrane system, thereby forming an aqueous mixture comprising a loaded micelle. The loaded micelle comprises the amphiphilic block copolymer in an amount of 85.0 wt. % to 99.9 wt. %, and the biologically active material in an amount of about 15.0 wt. % to 0.1 wt. %, each based on total dry weight of the loaded micelle. Preferably, the dialysis membrane has a molecular weight cutoff (MWCU) of about 1000 Da. Preferably, the amphiphilic block copolymer is biodegradable and/or biocompatible.
The membrane dialysis can be conducted with or without agitation. Smaller and more uniform nanoparticles of the loaded micelle are generally favored by less mechanical agitation (e.g., stirring). Preferably, dialysis is conducted using minimal or no agitation, accompanied by occasional replacement of the external water bath with fresh deionized water, allowing the water and organic solvent to exchange by diffusion alone. The resulting aqueous mixture can be centrifuged to remove large agglomerates. The resulting supernatant can contain about 37 wt. % to about 70 wt. % of the original combined dry weight of the block copolymer and the biologically active cargo material.
The dialysis is preferably conducted for a time period of 1 hour to 5 days, more preferably for a period of 1 day to 3 days.
The dialysis can be conducted at room temperature (18° C. to 28° C.), or at a lower temperature. In an embodiment, the dialysis is conducted at a temperature less than 10° C., more preferably 1° C. to 6° C. Lower temperature dialysis favors smaller loaded micelle particle sizes.
The term “loading efficiency” refers to the percentage of the initial weight of the biologically active material that is incorporated into the loaded micelle. The loading efficiency of the biologically active material in the loaded micelle is preferably at least 10%. Generally, the loading efficiency of the biologically active material is in a range of 10% to 50%, and even more specifically in a range of 30% to 50%.
Nanoparticles of the loaded micelle can have an average particle size (circular cross sectional diameter) of 10 nm to 500 nm, 10 nm to 250 nm, and preferably 25 nm to 200 nm as measured by dynamic light scattering. For the foregoing particle sizes, the aqueous solution can have a pH of 4.5 to 8.0, 5.0 to 7.0, or 6.0 to 7.0.
The organic solvent used to form a solution of the block copolymer and the biologically active cargo material is preferably miscible with water at concentrations of at least 1 microliter or more of organic solvent per 100 microliters of water. Exemplary organic solvents include methanol, ethanol, propanol, 2-propanol, 1-butanol, 2-butanol, t-butyl alcohol, acetone, 2-butanone, dimethoxyethane, diglyme, diethyl ether, methyl t-butyl ether, methylene chloride, ethyl acetate, ethylene glycol, glycerin, dimethylsulfoxide, dimethylformamide, acetic acid, tetrahydrofuran (THF), and dioxane.
As stated above, the biologically active cargo material can be a drug. Exemplary commercially available drugs include 13-cis-Retinoic Acid, 2-CdA, 2-Chlorodeoxyadenosine, 5-Azacitidine, 5-Fluorouracil, 5-FU, 6-Mercaptopurine, 6-MP, 6-TG, 6-Thioguanine, Abraxane, Accutane®, Actinomycin-D, Adriamycin®, Adrucil®, Afinitor®, Agrylin®, Ala-Cort®, Aldesleukin, Alemtuzumab, ALIMTA, Alitretinoin, Alkaban-AQ®, Alkeran®, All-transretinoic Acid, Alpha Interferon, Altretamine, Amethopterin, Amifostine, Aminoglutethimide, Anagrelide, Anandron®, Anastrozole, Arabinosylcytosine, Ara-C, Aranesp®, Aredia®, Arimidex®, Aromasin®, Arranon®, Arsenic Trioxide, Asparaginase, ATRA, Avastin®, Azacitidine, BCG, BCNU, Bendamustine, Bevacizumab, Bexarotene, BEXXAR®, Bicalutamide, BiCNU, Blenoxane®, Bleomycin, Bortezomib, Busulfan, Busulfex®, C225, Calcium Leucovorin, Campath®, Camptosar®, Camptothecin-11, Capecitabine, CaracTM, Carboplatin, Carmustine, Carmustine Wafer, Casodex®, CC-5013, CCI-779, CCNU, CDDP, CeeNU, Cerubidine®, Cetuximab, Chlorambucil, Cisplatin, Citrovorum Factor, Cladribine, Cortisone, Cosmegen®, CPT-11, Cyclophosphamide, Cyclosporin (an immunosuppressive agent, normally given to patients for life long after organ transplantation), Cytadren®, Cytarabine, Cytarabine Liposomal, Cytosar-U®, Cytoxan®, Dacarbazine, Dacogen, Dactinomycin, Darbepoetin Alfa, Dasatinib, Daunomycin, Daunorubicin, Daunorubicin Hydrochloride, Daunorubicin Liposomal, DaunoXome®, Decadron, Decitabine, Delta-Cortef®, Deltasone®, Denileukin Diftitox, DepoCytTM, Dexamethasone, Dexamethasone Acetate, Dexamethasone Sodium Phosphate Dexasone, Dexrazoxane, DHAD, DIC, Diodex, Docetaxel, Doxil®, Doxorubicin, Doxorubicin Liposomal, DroxiaTM, DTIC, DTIC-Dome®, Duralone®, Efudex®, EligardTM, EllenceTM, EloxatinTM, Elspar®, Emcyt®, Epirubicin, Epoetin Alfa, Erbitux, Erlotinib, Erwinia L-asparaginase, Estramustine, Ethyol, Etopophos®, Etoposide, Etoposide Phosphate, Eulexin®, Everolimus, Evista®, Exemestane, Fareston®, Faslodex®, Femara®, Filgrastim, Finasteride (for hair growth), Floxuridine, Fludara®, Fludarabine, Fluoroplex®, Fluorouracil, Fluorouracil (cream), Fluoxymesterone, Flutamide, Folinic Acid, FUDR®, Fulvestrant, G-CSF, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, Gemzar, GleevecTM, Gliadel® Wafer, GM-CSF, Goserelin, Granulocyte-Colony Stimulating Factor, Granulocyte Macrophage Colony Stimulating Factor, Halotestin®, Herceptin®, Hexadrol, Hexylen®, Hexamethylmelamine, HMM, Hycamtin®, Hydrea®, Hydrocort Acetate®, Hydrocortisone, Hydrocortisone Sodium Phosphate, Hydrocortisone Sodium Succinate, Hydrocortone Phosphate, Hydroxyurea, Ibritumomab, Ibritumomab Tiuxetan Idamycin®, Idarubicin, Ifex®, IFN-alpha Ifosfamide, IL-11 IL-2 Imatinib mesylate, Imidazole Carboxamide Interferon alfa, Interferon Alfa-2b (PEG Conjugate), Interleukin-2, Interleukin-11, Intron A® (interferon alfa-2b), Iressa®, Irinotecan, Isotretinoin, Ixabepilone, IxempraTM, K Kidrolase (t), Lanacort®, Lapatinib, L-asparaginase, LCR, Lenalidomide, Letrozole, Leucovorin, Leukeran, LeukineTM, Leuprolide, Leurocristine, LeustatinTM, Liposomal Ara-C, Liquid Pred®, Lomustine, L-PAM, L-Sarcolysin, Lupron®, Lupron Depot®, Matulane®, Maxidex, Mechlorethamine, Mechlorethamine Hydrochloride, Medralone®, Medrol®, Megace®, Megestrol, Megestrol Acetate, Melphalan, Mercaptopurine, Mesna, MesnexTM, Methotrexate, Methotrexate Sodium, Methylprednisolone, Meticorten®, Mitomycin, Mitomycin-C, Mitoxantrone, M-Prednisol®, MTC, MTX, Mustargen®, Mustine Mutamycin®, Myleran®, MylocelTM, Mylotarg®, Navelbine®, Nelarabine, Neosar®, NeulastaTM, Neumega®, Neupogen®, Nexavar®, Nilandron®, Nilutamide, Nipent®, Nitrogen Mustard, Novaldex®, Novantrone®, Octreotide, Octreotide acetate, Oncospar®, Oncovin®, Ontak®, OnxalTM, Oprevelkin, Orapred®, Orasone®, Oxaliplatin, Paclitaxel, Paclitaxel Protein-bound, Pamidronate, Panitumumab, Panretin®, Paraplatin®, Pediapred®, PEG Interferon, Pegaspargase, Pegfilgrastim, PEG-INTRONTM, PEG-L-asparaginase, PEMETREXED, Pentostatin, Phenylalanine Mustard, Platinol®, Platinol-AQ®, Prednisolone, Prednisone, Prelone®, Procarbazine, PROCRIT®, Proleukin®, Prolifeprospan 20 with Carmustine Implant, Purinethol®, Raloxifene, Revlimid®, Rheumatrex®, Rituxan®, Rituximab, Roferon-A® (Interferon Alfa-2a) Rubex®, Rubidomycin hydrochloride, Sandostatin®, Sandostatin LAR®, Sargramostim, Solu-Cortef®, Solu-Medrol®, Sorafenib, SPRYCELTM, Spironolactone, STI-571, Streptozocin, SU11248, Sunitinib, Sutent®, Tamoxifen, Tarceva®, Targretin®, Taxol®, Taxotere®, Temodar®, Temozolomide, Temsirolimus, Teniposide, TESPA, Thalidomide, Thalomid®, TheraCys®, Thioguanine, Thioguanine Tabloid®, Thiophosphoamide, Thioplex®, Thiotepa, TICE®, Toposar®, Topotecan, Toremifene, Torisel®, Tositumomab, Trastuzumab, Treanda®, Tretinoin, TrexallTM, Trisenox®, TSPA, TYKERB®, VCR, VectibixTM, Velban®, Velcade®, VePesid®, Vesanoid®, ViadurTM, Vidaza®, Vinblastine, Vinblastine Sulfate, Vincasar Pfs®, Vincristine, Vinorelbine, Vinorelbine tartrate, VLB, VM-26, Vorinostat, VP-16, Vumon®, Xeloda®, Zanosar®, ZevalinTM, Zinecard®, Zoladex®, Zoledronic acid, Zolinza, and Zometa.
Toxicity of the Loaded Micelle.
The toxicity of the loaded micelles toward a given cell can be at least comparable to the toxicity of the biologically active material alone toward the given cell. For example, a PTX loaded block copolymer can be as toxic to HepG2 as the PTX alone when tested under otherwise identical conditions.
INDUSTRIAL APPLICABILITY
Loaded micelles comprising a disclosed amphiphilic block copolymer and a biologically active material can be used for human and/or non-human therapeutic treatments. The compositions can be administered in the form of a powder, a pill, a liquid solution, paste, or a gel. The compositions can be used as a drug. The compositions can be administered orally or by way of other body cavities, by injection, intravenously, and/or topically. The compositions are particularly attractive for use in injectable systems for rigid, hydrophobic biologically active materials that have low water solubility, such as paclitaxel.
A method comprises contacting a cell with a loaded micelle, thereby killing the cell, wherein the loaded micelle comprises a disclosed amphiphilic block copolymer and a biologically active material. In another embodiment, the cell is a cancer cell, and the biologically active material is PTX.
An antimicrobial composition comprises a loaded micelle comprising a disclosed amphiphilic block copolymer and an antimicrobial material. The antimicrobial composition can be applied to a human and/or non-human animal tissue, including mammalian and/or non-mammalian animal tissue. The general term “animal tissue” includes wound tissue, burn tissue, skin, internal organ tissue, blood, bones, cartilage, teeth, hair, eyes, nasal surfaces, oral surfaces, other body cavity surfaces, and any cell membrane surfaces. In an embodiment, a method comprises contacting an animal tissue with a loaded micelle comprising the disclosed block copolymer and an antimicrobial material. In another embodiment, a method comprises contacting an animal tissue with an aqueous mixture of a loaded micelle comprising the disclosed amphiphilic block copolymer and an antimicrobial material. The antimicrobial compositions can be used in the form of a powder, a pill, or an aqueous mixture applied as a freely flowing liquid, spray, cream, injectable mixture, or gel. Uses include disinfectant washes for hands, skin, hair, bone, ear, eye, nose, throat, internal tissue, wounds, and teeth (e.g., as a mouthwash). Still other uses include disinfectants for articles such as medical devices. Medical devices include swabs, catheters, sutures, stents, bedpans, gloves, facial masks, absorbent pads, absorbent garments, internal absorbent devices, and insertable mechanical devices. In an embodiment, an article comprises a medical device in contact with the antimicrobial composition.
The antimicrobial compositions are also attractive as disinfecting agents for surfaces of articles (i.e., non-living articles) such as, for example, building surfaces in homes, businesses, and particularly hospitals. Exemplary home and commercial building surfaces include floors, door surfaces, bed surfaces, air conditioning surfaces, bathroom surfaces, railing surfaces, kitchen surfaces, and wall surfaces. Other articles include medical devices, cloths, garments, and non-medical equipment. Surfaces of articles can comprise materials such as wood, paper, metal, cloth, plastic, rubber, glass, paint, leather, or combinations thereof. In an embodiment, a method comprises contacting a surface of an article with the composition. In another embodiment, a method comprises contacting a surface of an article with an aqueous mixture of the composition.
The following examples demonstrate the preparation and use of loaded micelles prepared with an amphiphilic diblock copolymer produced by organocatalytic ring-opening polymerization of a steroidal monomer. The loaded micelles contain the anticancer drug paclitaxel.
EXAMPLES
Materials used in the following examples are listed in Table 7.









TABLE 7





ABBREVIATION
DESCRIPTION
SUPPLIER







TMC
Trimethylene Carbonate
Boehringer




Ingelheim


DBU
Diazabicyclo[5.4.0]Undec-7-Ene
Sigma




Aldrich


TU
N-Bis(3,5-
Prepared



Trifluoromethyl)Phenyl-N′-
below



Cyclohexylthiourea


Chlor-Cl
Cholesteryl Chloroformate
Sigma




Aldrich


mPEG-OH
Mono-Methyl Poly(Ethylene Glycol),
Polymer



number average molecular weight
Science



(Mn) 5K, degree of polymerization



DP ~113


Bis-MPA
2,2-Bis(Hydroxymethyl)Propionic
Sigma



Acid
Aldrich









Unless, specifically mentioned, all materials were purchased from Sigma-Aldrich or TCI. All solvents were of analytical grade, purchased from Fisher Scientific or J. T. Baker and used as received. Trimethylene carbonate (TMC) was purchased from Boehringer Ingelheim (Ingelheim, Germany), and dried extensively by freeze drying process under high vacuum. 1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) was distilled from CaH2 under dry N2 and transferred to glove box. Before transferring into glove box, monomers and other reagents (e.g., mPEG-OH) were dried extensively by freeze drying process under high vacuum.
N-bis(3,5-trifluoromethyl)phenyl-N′-cyclohexylthiourea (TU) was prepared as reported by R. C. Pratt, B. G. G. Lohmeijer, D. A. Long, P. N. P. Lundberg, A. Dove, H. Li, C. G. Wade, R. M. Waymouth, and J. L. Hedrick, Macromolecules, 2006, 39 (23), 7863-7871, and dried by stirring in dry THF over CaH2, filtering, and removing solvent under vacuum.
Nuclear Magnetic Resonance (NMR) Spectroscopy.
The 1H- and 13C-NMR spectra of monomers and polymers were recorded using a Bruker Avance 400 spectrometer, and operated at 400 and 100 MHz respectively, with the solvent proton signal as the internal reference standard.
Molecular Weight Determination by Size Exclusion Chromatography (SEC).
SEC was conducted using THF as the eluent for monitoring the polymer conversion and also for the determination of polystyrene equivalent molecular weights of the macro-transfer agents. THF-SEC was recorded on a Waters 2695D Separation Module equipped with a Waters 2414 differential refractometer and Waters HR-4E as well as HR-1 columns. The system was equilibrated at 30° C. in THF, which served as the polymer solvent and eluent with a flow rate of 1.0 mL/min. Polymer solutions were prepared at a known concentration (ca. 3 mg/mL) and an injection volume of 100 microliters was used. Data collection and analysis were performed using the Astra software (Wyatt Technology Corporation, USA; version 5.3.4.14). The columns were calibrated with a series of polystyrene standards ranging from Mp=360 Da to Mp=778 kDa (Polymer Standard Service, USA).
Monomer Synthesis.
Cholesteryl bearing cyclic carbonate monomer Chol-MTC was synthesized from commercially available cholesteryl chloroformate (Chol-Cl) according to Scheme 1.




The preparation involves three steps: 1) reaction of the choloroformate Chol-Cl with 2-bromoethyl amine hydrobromide in dichloromethane with triethylamine (TEA) to form carbamate Chol-Br; 2) base-catalyzed reaction of Chol-Br with the acid diol bis-MPA in dimethylformamide (DMF)/KOH to form the diol ester Chol-MPA, and 3) triphosgene-mediated cyclization of Chol-MPa to form the cyclic carbonate monomer Chol-MTC, with overall yield of about 26%. Detailed procedures of each of the three steps are provided below. 1H and 13C NMR were used to confirm the structures of the intermediates and the cyclic carbonate monomer.
Example 1
Preparation of Chol-Br. In a 500 mL round bottom flask, equipped with a magnetic stir bar, cholesterol chloroformate (25.0 g, 55.7 mmol, 1.0 equiv.) and 2-bromoethylamine hydrobromide (12.9 g, 63.0 mmol, 1.1 equiv.) were suspended in dichloromethane (200 mL) and the suspension was chilled in an ice-bath. To this suspension, a solution of triethylamine (TEA) (18.0 mL, 13.06 g, 129.1 mmol, 2.3 equiv.) in dichloromethane (100 mL) was added dropwise over 1 hour. The reaction mixture was maintained in the bath for an additional hour and was allowed to warm to room temperature. The reaction was then allowed to proceed for another 14 h, after which dichloromethane was removed under vacuo and the resultant solids were suspended in a 1:1 mixture of ethyl acetate and hexanes (300 mL). Organic layer was washed 2 times with a mixture of saturated brine (100 mL) and de-ionized water (50 mL), and one time with saturated brine (100 mL). The organic layer was dried over sodium sulfate and the solvents were removed under vacuo to yield a pale yellow solid (29.1 g, 97.4%). As the crude product was determined to have satisfactory purity by 1H NMR, no further purification was conducted. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.38 (CH═C in cholesterol), 5.03 (NHCOO of side-chain), 4.50 (CH—OCONH of cholesterol), 3.58 (BrCH2CH2NH), 2.45-0.6 (rest of the protons from cholesterol).
Example 2
Synthesis of Chol-MPA. In a 500 mL round bottom flask with magnetic stir bar, a mixture of KOH (85%, 2.0 g, 30.3 mmol, 1.1 equiv.), bis-MPA (4.20 g, 31.3 mmol, 1.1 equiv.) and dimethylformamide (DMF) (200 mL) were heated to 100° C. for 1.5 hours. A homogenous solution was formed, and Chol-Br (15.0 g, 28.0 mmol, 1.0 equiv.) was added to the hot solution. Stirring was continued with heating for 16 hours and most of the DMF was removed under reduced pressure, to result in oily semisolid, which was then dissolved in 2:1 ethyl acetate:hexanes mixture (300 mL). The organic solution was washed with saturated brine (100 mL) and de-ionized water (100 mL) mixture. The resultant aqueous layer was extracted with ethyl acetate (3×100 mL) to recover Chol-MPA lost during the washing process. The combined organic layers were washed with saturated brine (80 mL) and de-ionized water (20 mL) mixture. The combined organic layer was dried with Na2SO4 and the solvent removed in vacuuo to result in crude product as a pale white waxy solid (16.5 g). The crude product was purified by flash column chromatography using silica as the packing material and a gradient of hexanes to ethyl acetate as the eluent to result in the final product Chol-MPA as a waxy white solid (10.7 g, 64.8%). 1H NMR (400 MHz, CDCl3, delta, ppm): 5.35 (CH═C in cholesterol and NHCOO of side-chain), 4.47 (CH—OCONH of cholesterol), 4.26 (CH2CH2NHCOO), 3.88 and 3.72 (CH2OH) 3.45 (CH2CH2NHCOO), 3.34 (OH), 2.50-0.60 (rest of the protons from cholesterol and CH3 from bis-MPA).
Example 3
Preparation of Chol-MTC. In a 500 mL round bottom flask with magnetic stir bar, Chol-MPA (10.1 g, 17.1 mmol, 1.0 equiv.) was dissolved in anhydrous dichloromethane (150 mL). Pyridine (8.2 mL, 8.0 g, 101.5 mmol, 5.9 equiv.) was added and the solution was cooled in a dry ice-acetone bath (−78° C.). To this cooled reaction mixture, triphosgene (2.69 g, 9.06 mmol, 1.9 equivalents based on functional equivalents of triphosgene) solution (dissolved in 50 mL dichloromethane) was added dropwise over 1 hour. After 1 hour, from −78° C., the reaction mixture was allowed to warm up to room temperature, and after 2 hours, the reaction was quenched by adding saturated aqueous ammonium chloride solution (50 mL). The organic layer was washed twice with a mixture of 1.0 N HCl (20 mL) and saturated brine (80 mL), followed by a mixture of saturated brine (50 mL) and saturated NaHCO3 (50 mL), dried using Na2SO4. Removal of solvent in vacuo resulted in crude product as a slightly yellowish solid. The crude product was further purified by flash column chromatography using silica as the packing material and a gradient of chloroform to chloroform:ethyl acetate(4:1) mixtures as the eluent, to result in the final product Chol-MTC as a waxy white solid (6.8 g, 65%). 1H NMR (400 MHz, CDCl3, delta, ppm): 5.35 (CH═C in cholesterol), 4.95 (NHCOO), 4.86 and 4.27 (CH2OCOOCH2), 4.47 (CH—OCONH of cholesterol), 4.27 (CH2CH2NHCOO), 3.45 (CH2CH2NHCOO), 2.40-0.60 (rest of the protons from cholesterol and CH3 in the cyclic carbonate monomer).
Ring Opening Polymerizations.
The ring opening homopolymerization of Chol-MTC resulted in low conversion and/or low degree of polymerization when the ring opening polymerization was initiated by benzyl alcohol (BzOH) or mPEG-OH. On the other hand, copolymerization of Chol-MTC with TMC was found to occur with high conversion (>90%). Therefore, TMC was used as a diluent comonomer to prepare several amphiphilic block copolymers according to Scheme 2.







The TMC not only assists in achieving high monomer conversions, but also serves to dilute/spread out the rigid cholesteryl side chain groups across the hydrophobic region. The cholesteryl bearing polymers prepared according to Scheme 2 are represented by the name Polymer(x:y), where x and y represent numbers of units derived from Chol-MTC and TMC, respectively, in the block copolymer structure of Scheme 2. The subscript n in the mPEG-OH structure (Scheme 2) was about 113, corresponding to a number average molecular weight (Mn) of 5000 Da. The stacked subunit structures between the vertical brackets in the block copolymer structure of Scheme 2 indicate the polycarbonate block of Polymer(x:y) is a random copolymer of repeat units derived from Chol-MTC and TMC.
In the following Examples 4 to 9, amounts were calculated based on following: i) the initiator (mPEG-OH) has one nucleophilic initiating group (hydroxy group) per mole of the initiator, ii) the molecular weight of mPEG-OH=5000 g/mole, and iii) the molar amount of initiator was set to one equivalent.
Example 4
Preparation of Polymer(0:67). In a 7 mL vial containing a magnetic stir bar, in glove box, TMC (MW=102.1, 922 mg, 9.03 mmol, 180.6 equivalents), mPEG-OH (5 kDa, 250 mg, 50.0 micromoles, 1.0 equivalent) and TU (MW=370.4, 47.0 mg, 127 micromoles, 2.5 equiv.) were dissolved in dichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7 microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and size exclusion chromatography (SEC). After 40 hours, the reaction was quenched by the addition of about 20 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer(0:67) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (580 mg). The number average molecular weight by NMR (Mn (NMR))=11800 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 4.20-4.30 (CH2CH2CH2OCOO), 3.85-3.5, 3.38 (CH3 of mPEG), 2.10-2.00 (CH2CH2CH2OCOO). The degree of polymerization (DP) of TMC was 67. The polydispersity index (PDI) was 1.12.
Example 5
Preparation of Polymer(4:0). In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 312 mg, 507 micromoles, 10.1 equivalents), mPEG-OH (5 kDa, 251 mg, 50.2 micromoles, 1.0 equivalent) and TU (MW=370.4, 46.0 mg, 125 micromoles, 2.5 equiv.) were dissolved in dichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7 microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 18 hours, the reaction was quenched by the addition of about 20 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (4:0) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (356 mg). Mn (NMR)=7500 Da. 1H NMR (400 MHz, CDCl3, 6, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2 and CH2CH2NHCOO), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol and CH3 in the cyclic carbonate monomer). The DP of Chol-MTC=4. PDI=1.12.
Example 6
Preparation of Polymer(11:0). In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 312 mg, 995 micromoles, 19.1 equivalents), mPEG-OH (5 kDa, 260 mg, 52.0 micromoles, 1.0 equivalent) and TU (MW=370.4, 48.0 mg, 130 micromoles, 2.5 equiv.) were dissolved in dichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7 microliters, 19.1 mg, 124 micromoles, 2.4 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 6 hours, the reaction was quenched by the addition of about 20 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (11:0) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (612 mg). Mn (NMR)=11800 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2 and CH2CH2NHCOO), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol and CH3 in the cyclic carbonate monomer). The DP of Chol-MTC=11. PDI=1.21.
Ring opened block copolymers having different x:y ratios (Chol-MTC units:TMC units) were prepared using the initiator .mPEG-OH (5.0 kDa).
Example 7
Preparation of Polymer (8:8). In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 617 mg, 1000 micromoles, 9.9 equivalents), TMC (MW=102.1, 109 mg, 1.07 mmol, 10.6 equivalents), mPEG-OH (5 kDa, 506 mg, 101 micromoles, 1.0 equivalent) and TU (MW=370.4, 103 mg, 278 micromoles, 2.7 equiv.) were dissolved in dichloromethane (2.0 mL). To this solution, DBU (MW=152.24, 37.3 microliters, 38.0 mg, 250 micromoles, 2.5 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 5 hours, the reaction was quenched by the addition of about 30 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (8:8) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (968 mg). Mn (NMR)=10700 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2, CH2CH2NHCOO and CH2CH2CH2OCOO of TMC), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol, CH3 in the cyclic carbonate monomer and CH2CH2NHCOO of TMC). The DP of Chol-MTC=8. The DP of TMC=8. PDI=1.18.
Example 8
Preparation of Polymer(11:30). In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 699 mg, 520 micromoles, 10.4 equivalents), TMC (MW=102.1, 154 mg, 1.51 mmol, 30.3 equivalents), mPEG-OH (5 kDa, 249 mg, 49.8 micromoles, 1.0 equivalent) and TU (MW=370.4, 46.0 mg, 125 micromoles, 2.5 equiv.) were dissolved in dichloromethane (2.5 mL). To this solution, DBU (MW=152.24, 18.7 microliters, 19.1 mg, 124 micromoles, 2.5 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 18 hours, the reaction was quenched by the addition of about 20 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (11:30) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (614 mg). Mn (NMR)=14800 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2, CH2CH2NHCOO and CH2CH2CH2OCOO of TMC), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol, CH3 in the cyclic carbonate monomer and CH2CH2NHCOO of TMC). DP of Chol-MTC=11. The DP of TMC=30. PDI=1.20.
Examples 9
Preparation of Polymer(18:55). In this example, a higher level of catalyst (5.0 equiv of with respect to initiator) was used because it was found from Example 4 that polymerization kinetics were slower for relatively high initiator to monomer ratios. In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 876 mg, 1.42 mmol, 19.6 equivalents), TMC (MW=102.1, 452 mg, 4.43 mmol, 61.2 equivalents), mPEG-OH (5 kDa, 362 mg, 72.4 micromoles, 1.0 equivalent) and TU (MW=370.4, 134 mg, 362 micromoles, 4.8 equiv.) were dissolved in dichloromethane (3.0 mL). To this solution, DBU (MW=152.24, 52.3 microliters, 53.3 mg, 350 micromoles, 5.0 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 4 hours, the reaction was quenched by the addition of about 30 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (18:55) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (533 mg). Mn (NMR)=21700 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2, CH2CH2NHCOO and CH2CH2CH2OCOO of TMC), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol, CH3 in the cyclic carbonate monomer and CH2CH2NHCOO of TMC). DP of Chol-MTC=18. The DP of TMC=55. PDI=1.17.
Example 10
Preparation of Polymer(29:77). In a 7 mL vial containing a magnetic stir bar, in glove box, Chol-MTC (MW=616, 946 mg, 1.54 mmoles, 30.0 equivalents), TMC (MW=102.1, 464 mg, 4.55 mmol, 92.4 equivalents), mPEG-OH (5 kDa, 256 mg, 51.2 micromoles, 1.0 equivalent) and TU (MW=370.4, 95.0 mg, 257 micromoles, 4.9 equiv.) were dissolved in dichloromethane (3.0 mL). To this solution, DBU (MW=152.24, 37.3 microliters, 38 mg, 250 micromoles, 5.0 equivalents) was added to initiate polymerization. The reaction mixture was allowed to stir at room temperature and aliquots of samples were taken to monitor the monomer conversion and the molecular weight by 1H NMR spectroscopy and SEC. After 4 hours, the reaction was quenched by the addition of about 30 mg of benzoic acid and was precipitated in ice-cold diethyl ether (2×50 mL). Polymer (29:77) was dried in a tared vial for about 1 to 2 days, until a constant sample mass was obtained as white power (590 mg). Mn (NMR)=30700 Da. 1H NMR (400 MHz, CDCl3, delta, ppm): 5.45-5.35 (CH═C in cholesterol), 5.35-5.05 (NHCOO of side-chain), 4.65-4.40 (CH—OCONH of cholesterol), 4.40-4.10 (CH2OCOOCH2, CH2CH2NHCOO and CH2CH2CH2OCOO of TMC), 3.85-3.5 (CH2CH2O of mPEG) 3.5-3.30 (CH2CH2NHCOO), 3.38 (CH3 of mPEG), 2.45-0.55 (rest of the protons from cholesterol, CH3 in the cyclic carbonate monomer and CH2CH2NHCOO of TMC). DP of Chol-MTC=29. The DP of TMC=77. PDI=1.21.
Each of the above described block copolymers has a polydispersity index (PDI) of less than 1.25.
Properties of the self-assembled polymers without drug loading are summarized in Table 8. Example 4 and Examples 7 to 10 have an x:y ratio less than or equal to 1.0 (x:y=0:67, 8:8, 11:30; 18:55; 29:77, respectively). Examples 5 and 6 have an x:y ratio greater than 1.0 (x:y=4:0, 11:0, respectively).














TABLE 8







Units of
Units of


Micelle





Chol-MTC
TMC

CMC
Size


Example
Polymer (x:y)
(x)
(y)
Mn
(mg/L)
(nm)
Micelle PDI






















4
Polymer (0:67)
0
67
11800
2.1
163 ± 4
0.56 ± 0.01


5
Polymer (4:0)
4
0
7500
—
 234.5 ± 33.0
0.49 ± 0.00


6
Polymer (11:0)
11
0
11600
2.1
114 ± 1
0.14 ± 0.01


7
Polymer (8:8)
8
8
10700
1.5
133 ± 3
0.44 ± 0.01


8
Polymer (11:30)
11
30
14800
1.5
 36 ± 1
0.08 ± 0.05


9
Polymer (18:55)
18
55
21700
2.1
 229 ± 51
0.29 ± 0.02


10
Polymer (29:77)a
29
77
30700





aExample 10 was not further characterized or used for preparing loaded micelles below due to its hydrophobicity.






Critical Micelle Concentrations.

General procedure for polymer self-assembly. The following dialysis method was used to prepare non-loaded micelles of the polymers of Examples 4 to 9. In a scintillation vial (20 mL) with a magnetic stir bar, block copolymer (15.0 mg) was dissolved in DMF (2.0 mL) with stirring at room temperature for about 1 hour to 2 hours. The polymer solution in DMF was then transferred to a prewashed dialysis membrane having a molecular weight cutoff (MWCO) of 1000 Da (Spectra/Por) and was dialyzed at 4° C. against deionized water (1 L). The water was changed at 3, 6 and 24 hours. At the end of the dialysis process, the resulting micelle solution was centrifuged at 4000 rpm for 5 minutes to remove large aggregates. Typically the final concentration of the polymer solution after dialysis was about 1.0 mg/mL.
Fluorescence Measurements. The critical micelle concentrations (CMCs) of the polymers in DI water were determined by fluorescence spectroscopy using pyrene as the probe. The fluorescence spectra were recorded by an LS 50B luminescence spectrometer (Perkin Elmer, U.S.A.) at 25° C. Dialyzed polymer samples (see below) were equilibrated for 10 min before taking measurements. Aliquots of pyrene in acetone solution (6.16×10−5 M, 10 microliters) were added to glass vials and air dried to remove the acetone. Polymer solutions of varying concentrations were added to the pyrene at 1 mL each, and left to stand for 24 hours. The final pyrene concentration in each vial is 6.16×10−7 M. The excitation spectra were scanned at wavelength from 300 nm to 360 nm with an emission wavelength of 395 nm. Both the excitation and emission bandwidths were set at 2.5 nm. The intensity (peak height) ratio of I339/I334 from the excitation spectra was analyzed as a function of polymer concentration. The CMC was taken at the point of intersection between the tangent to the curve at the inflection and tangent of the points at low concentrations.
Transmission electron microscopy. The morphologies of the polymers under aqueous conditions were observed under a FEI Tecnai G2 F20 electron microscope using an acceleration voltage of 200 keV. The TEM samples were prepared by first placing a drop of aqueous polymer solution (4.0 microliters) onto a formvar coated 200 mesh copper grid (Ted Pella Inc., USA). After 1 min, the excess solution was wicked off by using filter paper. Then the staining agent of phosphotungstic acid (2% w/v; 4.0 microliters) was placed on the grid and after a minute, the excess solution was wicked off and the grid was left to dry under ambient conditions.
FIG. 1 is a graph showing the intensity (peak height) ratio of I339/I334 of the excitation spectra as a function of polymer concentration for Example 4 (x:y=0:67), Example 6 (x:y=11:0), Example 7 (x:y=8:8), Example 8 (x:y=11:30), and Example 9 (x:y=18:55). The CMC values in DI water of Examples 4, and 6 to 9 are, respectively, 2.1 mg/L, 2.1 mg/L, 1.5 mg/L, 1.5 mg/L and 2.1 mg/L (Table 8). The CMC values of the polymers of Example 5 and Example 10 were not determined. The low CMC values of Examples 6 to 8 indicate that the micellar structure can exist even at low concentrations, thereby enabling in vivo application of these materials where the micellar solution would undergo an extensive dilution after administration. The polymers are expected to exhibit even lower CMC values in the bloodstream as a result of the “salting out” effect.
The average particle size diameter (in nanometers) and size distribution (PDI) of non-loaded micelles formed with the polymers are also listed in Table 8. Example 8, which has an x:y ratio (Chol-MTC:TMC=11:30) less than 1.0 and a molecular weight less than or equal to 15 kDa, the average particle size was less than 40 nm. This value is significantly smaller than the micelle particle sizes obtained with Example 5 (163 nm), Example 6 (114 nm), and Example 7 (133 nm), which have an x:y ratio greater than or equal to 1.0. The micelle polydispersity index (PDI) also indicates that the nanoparticles formed from the polymers having an x:y ratio less than 1:1 and lower molecular weight of about 15 kDa (Examples 8) have a lower particle size distribution compared to the polymers having an x:y ratio greater than or equal to about 1.0 (Examples 5 to 7).
FIGS. 2A to 2F are TEM images of the micelles formed by Example 4 (x:y=0:67), Example 5 (x:y=4:0), Example 6 (x:y=11:0), Example 7 (x:y=8:8), Example 8 (x:y=11:30), and Example 9 (x:y=18:55), respectively. Comparing the TEMs of Examples 4 to 6 (FIGS. 2A to 2C) self-assembly was promoted by increasing the content of cholesteryl repeat units in the block copolymer. When TMC was used as a comonomer, discrete nanostructures were observed (Examples 8 and 9, FIGS. 2D and 2E, respectively). At high TMC content, predominantly collapsed vesicles formed (Example 9, FIG. 2F). The upper image in FIG. 2F is at a magnification of 28000×. The lower image in FIG. 2F is at a magnification of 110000×.
Cytotoxicity of Amphiphilic Block Copolymers.
HepG2 cells were maintained in DMEM growth medium supplemented with 10% FBS (fetal bovine serum), 100 microgram/mL penicillin and 100 units/mL streptomycin at 37° C., under the atmosphere of 5% CO2. To assess the cytotoxicity of the amphiphilic block copolymer (Examples 4, and Examples 6 to 9) in HepG2 cells, a standard MTT (dimethyl thiazolyl diphenyl tetrazolium salt) assay protocol was employed. On a 96-well plate, cells were seeded at a density of 1×104 cells/well and allowed to grow for 24 hours to reach 60% to 70% confluence. Each well was replaced with 100 microliters of fresh growth medium and treated with 10 microliters of the block copolymer solution. The cytotoxicity test was performed in replicates of 6 wells per block copolymer or PTX concentration. After 4 hours of incubation, the wells were replaced with fresh medium and incubated further for 68 hours. Upon replacing the wells with 100 microliters of fresh medium and 20 microliters of MTT solution (5 mg/mL in PBS buffer), the cells were incubated for another 4 hours. Finally, the used media were removed and the internalized purple formazan crystals in each well were dissolved with 150 microliters of DMSO. A 100 microliter aliquot of the formazan/DMSO solution was transferred from each well to a new 96-well plate, and the absorbance (A) was measured using a microplate spectrophotometer (BioTek Instruments Inc, Winooski, Vt., U.S.A.) at the wavelength of 550 nm and 690 nm. To measure the relative cell viability in different polymer, the absorbance of formazan solution in the treated cells were compared to that of the control cells:

Cell viability=[(A550−A690)sample/(A550−A690)control]×100%

where A550 and A690 represent absorbance at 550 nm and 690 nm, respectively, (A550−A690) sample represents the difference in the absorbance at 550 nm and 690 nm of the sample, and (A550−A690) control represents the difference in the absorbance at 550 nm and 690 nm for the control.

The data were statistically analyzed for significant differences, based on the Student's t-test at p<0.05. FIG. 3 is a graph showing HepG2 cell viability (%) against block copolymer concentration (Examples 4, and 6 to 9) without the drug. Each of the block copolymers was non-cytotoxic. Greater than 90% cell viability was observed even at the highest polymer concentration (2377 mg/L).
Preparation of Drug Loaded Micelles.
The following examples demonstrate the following drugs can be physically entrapped by non-covalent interactions in the self-assembled block copolymer: Paclitaxel (PTX), Cyclosporin A (CYC, an immunosuppressive agent used after organ transplantation) and Spironolactone (SPL, a drug used for hair growth).
General procedure for all three drugs (PTX, CYC and SPL). The dialysis method was used to prepare loaded micelles with the drugs. Polymer (15 mg) and drug (3 mg) were dissolved together in 2 mL DMF in their respective ratios. The mixture was placed in a dialysis membrane bag with molecular weight cut-off (MWCO) of 1000 Da (Spectrum Laboratories, U.S.A.). The dialysis bag was then immersed in 1 L of deionized (DI) water at 4° C. without stirring for 2 days. During the 2-day course, the external dialysis medium was replaced at 3 hours, 6 hours and 24 hours. At the end of the dialysis process, the resulting micelle solution was centrifuged at 4000 rpm for 5 minutes to remove large agglomerates. The dry weight of the agglomerates was about 30 wt. % to about 60 wt. % of the combined dry weight of the polymer and drug. The loaded micelles were characterized with respect to their size with a Zetasizer with dynamic light scattering capability (scattering angle: 90°) and equipped with a He—Ne laser beam at 658 nm (Malvern Instruments Zetasizer Nano ZS, UK).
PTX loading measurements. To determine the encapsulation efficiency and loading level of PTX, the drug-loaded micelles were first freeze-dried and then re-dissolved in 200 microliters of dichloromethane. 6 mL of cold ether was then added to precipitate the polymer. The mixture was centrifuged at 4000 rpm for 20 minutes and the supernatant was transferred into a fresh tube, and air dried. The deposited drug was dissolved in 4 mL of mobile phase consisting of water:methanol:acetonitrile in the volume ratio of 35:20:45, and filtered through a 0.22 micrometer filter to remove any large aggregates. PTX loading level as weight percent (wt. %) based on the total weight of the dry loaded micelle was analyzed using high performance liquid chromatography (HPLC, Waters 996 PDA detector, U.S.A.) at 228 nm. The drug loading efficiency (i.e., the percentage of the initial amount in milligrams of drug that is successfully encapsulated) was also reported. For example, if the loading efficiency is 40%, then 40% of the initial amount of paclitaxel was incorporated into the loaded micelles obtained after freeze-drying.
CYC loading measurements. To determine the encapsulation efficiency and loading level of CYC, the drug-loaded micelles were first freeze-dried and then re-dissolved in 200 microliters of dichloromethane. 6 mL of cold ether was then added to precipitate the polymer. The mixture was centrifuged at 4000 rpm for 20 minutes and the supernatant was transferred into a fresh tube, and air dried. The deposited drug was dissolved in 4 mL of mobile phase consisting of water:acetonitrile in the volume ratio of 20:80. CYC loading level as weight percent (wt. %) based on the total weight of the drug-loaded micelle was analyzed using high performance liquid chromatography (HPLC, Waters 996 PDA detector, U.S.A.) at 210 nm.
SPL loading measurements. To determine the encapsulation efficiency and loading level of SPL, the drug-loaded micelles were first freeze-dried and then re-dissolved in 200 microliters of dichloromethane. 6 mL of cold ether was then added to precipitate the polymer. The mixture was centrifuged at 4000 rpm for 20 minutes and the supernatant was transferred into a fresh tube, and air dried. The deposited drug was dissolved in 4 mL of mobile phase consisting of water:acetonitrile in the volume ratio of 50:50. SPL loading level as weight percent (wt. %) based on the total weight of the drug-loaded micelle was analyzed using high performance liquid chromatography (HPLC, Waters 996 PDA detector, U.S.A.) at 238 nm.
Examples 11 to 16
Using the above general procedure, drug loaded micelles were prepared using the polymers of Examples 4, and 6 to 10 by dialyzing at 4° C. without stirring. The loaded micelles are designated by the name PTX-Polymer(x:y), CYC-Polymer(x:y) and SPL-Polymer(x:y), where x is the number of repeat units derived from Chol-MTC and y is the number of repeat units derived from TMC.
The effects of block copolymer structure and initial drug loading on the particle size, polydispersity index (PDI), final drug loading level, and loading efficiency of the PTX loaded micelles are shown in Tables 9, 10 and 11, respectively.















TABLE 9








Units










of
Units
Loaded





Chol-
of
Micelle
Loaded
Loading
Loading




Initial PTX
MTC
TMC
Size
Micelle
level
efficiency


Example
Loaded Micelle
(mg)
(x)
(y)
(nm)
PDI
(wt. %)
(%)























11
PTX-
3
0
67
142 ± 52
0.49 ± 0.17
5.0 ± 2.7
27.6 ± 7.8



Polymer (0:67)


12
PTX-
3
11
0
115 ± 9
0.13 ± 0.03
3.8 ± 0.3
23.3 ± 3.6



Polymer (11:0)


13
PTX-
3
8
8
131 ± 9
0.45 ± 0.08
9.2 ± 0.8
38.8 ± 5.0



Polymer (8:8)


14
PTX-
3
11
30
 36 ± 1
0.07 ± 0.01
15.0 ± 1.8 
56.8 ± 0.3



Polymer (11:30)


15
PTX-
3
18
55
200 ± 44
0.22 ± 0.03
8.4 ± 2.4
25.5 ± 0.3



Polymer (18:55)
























TABLE 10








Units










of
Units
Loaded




Initial
Chol-
of
Micelle
Loaded
Loading
Loading




CYC
MTC
TMC
Size
Micelle
level
efficiency


Example
Loaded Micelle
(mg)
(x)
(y)
(nm)
PDI
(wt. %)
(%)























16
CYC-
3
0
30
125 ± 18
0.39 ± 0.02
 7.9 ± 1.6
31.9 ± 3.8



Polymer (0:30)


17
CYC-
3
11
0
123 ± 0.3
0.20 ± 0.01
12.7 ± 0.4
51.3 ± 4.7



Polymer (11:0)


18
CYC-
3
11
30
 36 ± 0.2
0.04 ± 0.01
16.8 ± 2.6
60.6 ± 3.7



Polymer (11:30)
























TABLE 11








Units










of
Units
Loaded




Initial
Chol-
of
Micelle
Loaded
Loading
Loading




CYC
MTC
TMC
Size
Micelle
level
efficiency


Example
Loaded Micelle
(mg)
(x)
(y)
(nm)
PDI
(wt. %)
(%)























19
SPL-
3
0
30
112 ± 24
0.43 ± 0.01
3.7 ± 0.3
11.9 ± 1.6



Polymer (0:30)


20
SPL-
3
11
0
102 ± 2
0.16 ± 0.01
4.3 ± 0.2
14.3 ± 0.9



Polymer (11:0)


21
SPL-
3
11
30
 37 ± 1
0.08 ± 0.02
4.9 ± 1.1
14.4 ± 6.4



Polymer (11:30)









Overall, an initial amount of PTX of 3 mg produced loaded micelles having a particle size (i.e., average circular cross-sectional diameter) in a range of 36 nm to 200 nm (Examples 11 to 15, Table 9). The optimization studies of the fabrication conditions for the loaded micelle show that stirring conditions had less influence on the particle size than the temperature at which the loaded micelles were prepared. Hence, the particles were formed solely based on slow diffusion and solvent exchange. Particle sizes less than 200 nm are attractive for drug delivery because the particles are less susceptible to clearance by the reticuloendothelial system (RES), and have a more favorable biodistribution into tumor tissues through the enhanced permeability and retention (EPR) effect. This passive targeting phenomenon is attributed to two factors: the disorganized and leaky tumor vasculature which leads to hyperpermeability to macromolecules, and at the same time, tumor tissues have poor lymphatic drainage, which prevents escape of these macromolecules after entry. In greater detail, tumor vessels are highly disorganized, defective or leaky having gap sizes of 100 nm to 2 micrometers. This allows nanoparticles to escape easily from the blood stream and accumulate within tumors. Furthermore, re-entry back into the bloodstream is prevented by the defective lymphatic drainage in the tumor.
Under the same fabrication conditions, copolymers prepared from a mixture of Chol-MTC and TMC (Examples 13 to 15, Table 9) had significantly higher drug loading capacities compared to the loading capacities of the block copolymer formed with TMC alone (Example 11, PTX-Polymer(0:67)), or the block copolymer prepared with Chol-MTC alone (Example 12, PTX-Polymer(11:0)). The combination of TMC and Chol-MTC in the block copolymer provided increased loading efficiency (%), especially at higher x:y ratios (compare Examples 11 and 12 to Examples 13 and 14, Table 9). Lower PTX loading level (wt. %), lower loading efficiency (%), and larger particle size were observed with Example 15 (PTX-Polymer(18:55)), attributed to precipitation of the polymer and/or PTX during the dialysis process.
FIG. 4A to FIG. 4E are TEM images of loaded micelles formed by Example 11 (PTX-Polymer(0:67), FIG. 4A), Example 12 (PTX-Polymer(11:0), FIG. 4B), Example 13 (PTX-Polymer(8:8), FIG. 4C), Example 14 (PTX-Polymer(11:30), FIG. 4D), and Example 15 (PTX-Polymer(18:55), FIG. 4E), respectively. When Chol-MTC was used alone in the ring opening polymerization (Example 12, FIG. 4B), the resulting block copolymer self-assembled into cylindrical columns about 20 nanometers wide. The columns comprised a stack of discs, each disc having a height of about 5 nanometers; thus, a stack of 9 discs corresponds to a cylindrical column length of about 45 nanometers. When Chol-MTC was copolymerized with TMC, discrete spherical nanostructures were formed (Examples 13 to 15, FIGS. 4C to 4E, respectively) having an average diameter of about 50 nm to about 100 nm.
In Vitro Release Studies.
Release of drugs from the drug-loaded nanoparticles (Examples 11 to 15) was studied using the dialysis method. A dialysis membrane tube with MWCO of 2000 Da (Spectrum Laboratories, U.S.A.) containing 2 ml of the micelles was immersed in 40 ml of the release medium (i.e., PBS at pH 7.4) containing 0.1% (v/v) TWEEN 80 (sold by ICI Americas, Inc.) to maintain a sink condition. This was kept shaking on an orbital shaker at 120 rpm at 37° C. At designated time intervals (1, 7, 24, 31, 48, 55, 120 and 144 hours), the release medium was removed and replaced with fresh medium. The collected medium was analyzed for its drug content. To do this, 10 ml of dichloromethane (DCM) was added and mixed with the release medium through 3 minutes of vigorous mixing. The organic layer was allowed to settle and was carefully extracted into a new vial. DCM was evaporated by air flow. The deposited drug was dissolved in 4 ml of mobile phase consisting of 20 mM ammonium acetate in water:methanol:acetonitrile in the volume ratio of 35:20:45. Drug content was analyzed using high performance liquid chromatography (HPLC, Waters 996 PDA detector, U.S.A.) at 228 nm UV wavelength.
FIGS. 5A to 5C are graphs showing the cumulative release of PTX, CYC and SPL, respectively, with time. Approximately 100% of the PTX was released from PTX-Polymer(11:0) micelles (Example 12, FIG. 5A) within 24 hours. About 60% of the PTX was released by 48 hours, and was sustained for more than 6 days from the remaining PTX loaded micelles (Examples 11, 13, 14 and 15, FIG. 5A). For CYC, drug release was sustained from the micellar formulation (CYC-Polymer(11:30), Example 18) for about 5 days (FIG. 5B). SPL release from the micellar formulation (SPL-Polymer(11:30), Example 21) was sustained for about 7 hours (FIG. 5C).
Killing Efficiency of Tumor Cells by PTX-Loaded Micelles.
Killing efficiency of HepG2 cells by PTX-loaded micelles (Examples 11 to 15) was studied according to the protocol described above for the block copolymers alone. FIG. 6 is a bar chart comparing viability of HepG2 cells after incubation for 48 hours with PTX loaded micelles (Examples 11 to 15) and free PTX (PTX not bound to a polymer) at various PTX concentrations. The PTX loaded micelles had better killing efficiency than the free PTX alone at all PTX levels. As described above (see also FIG. 3), the polymers alone were not cytotoxic against HepG2 cells. Therefore, the decrease in cell viability after 48 hours incubation with PTX or PTX-loaded micelles can be attributed to PTX released from the micelles. The loaded micelle PTX-Polymer(8:8) (Example 13) killed HepG2 cells more efficiently than the free PTX at most PTX concentrations. A similar trend was observed with loaded micelle PTX-Polymer(11:0) (Example 12) at low PTX concentrations. The killing efficiency of loaded micelles PTX-Polymer(0:67) (Example 11), PTX-Polymer(11:30) (Example 14), and PTX-Polymer(18:55) (Example 15) against HepG2 cells was greater than free PTX at all concentrations tested. These findings show that a PTX-loaded nanoparticulate formulation was more efficient than free PTX in killing HepG2 cells.
The above examples demonstrate that hydrophobic cholesterol-functionalized cyclic carbonate monomers, such as Chol-MTC, allow for efficient incorporation of cholesterol groups into a copolymers via metal-free organocatalytic ring opening polymerization, particularly when copolymerized with a diluent cyclic carbonyl monomer such as TMC. Amphiphilic, biocompatible and/or biodegradable block copolymers can be formed using a mono-endcapped poly(alkylene glycol) initiators for the ring opening polymerization. The cholesterol functionalized block copolymers have low CMCs, and have high loading capacity for extremely hydrophobic and/or rigid drugs. The encapsulation of the drug can be accomplished via self-assembly without sonication or homogenization using the above described dialysis techniques. Consequently, these polymeric materials are promising nanocarriers for delivery of hydrophobic anticancer drugs such as PTX and other drugs with similar ring structures such as CYC and SPL.
The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms “comprises” and/or “comprising,” when used in this specification, specify the presence of stated features, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and/or groups thereof. When a range is used to express a possible value using two numerical limits X and Y (e.g., a concentration of X ppm o Y ppm), unless otherwise stated the value can be X, Y, or any number between X and Y.
The description of the present invention has been presented for purposes of illustration and description, but is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the invention. The embodiments were chosen and described in order to best explain the principles of the invention and their practical application, and to enable others of ordinary skill in the art to understand the invention.

## Claims

1. An amphiphilic block copolymer, comprising:
a hydrophilic poly(alkylene oxide) block; and
a biodegradable hydrophobic block comprising a first repeat unit of formula (3):





wherein i) t and t′ are integers independently having a value of 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, iv) S′ is a steroidal group.

2. The block copolymer of claim 1, wherein t and t′ are each 1, each Q1 at carbon 4 is hydrogen, each Q1 at carbon 6 is hydrogen, and Q1 at carbon 5 is selected from the group consisting of hydrogen, methyl, and ethyl.

3. The block copolymer of claim 1, wherein S′ is a cholesteryl group:

4. The block copolymer of claim 1, wherein the hydrophobic block comprises a second repeat unit comprising a backbone carbonate group, and the second repeat unit has no steroidal group.

5. The block copolymer of claim 1, wherein the hydrophobic block comprises a second repeat unit comprising a backbone ester group.

6. The block copolymer of claim 5, wherein the hydrophilic block comprises a poly(ethylene oxide) chain segment, and the second repeat unit has no steroidal group.

7. The block copolymer of claim 1, wherein the block copolymer has a critical micelle concentration of 1 mg/L to 50 mg/L.

8. The block copolymer of claim 1, wherein a bond joining L′ and S′ is hydrolytically and/or enzymatically cleavable.

9. The block copolymer of claim 1, wherein L′ has the formula (10):




wherein L′′′ is a divalent linking group comprising 1 to 20 carbons.

10. A loaded micelle comprising:
85.0 wt. % to 99.9 wt. % of the amphiphilic block copolymer comprising a) a hydrophilic poly(alkylene oxide) block and b) a biodegradable hydrophobic block comprising a carbonate repeat unit of formula (3):





wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group; and
15 wt. % to 0.1 wt. % of a biologically active cargo material bound by non-covalent interactions to the block copolymer, wherein weight percent (wt. %) is based on total dry weight of the loaded micelle.

11. The loaded micelle of claim 10, wherein the loaded micelle has an average cross-sectional diameter of 25 nm to 200 nm.

12. The loaded micelle of claim 10, wherein t and t′ are each 1, each Q1 at carbon 4 is hydrogen, each Q1 at carbon 6 is hydrogen, and Q1 at carbon 5 is selected from the group consisting of hydrogen, methyl, and ethyl.

13. The loaded micelle of claim 10, wherein S′ is a cholesteryl group:

14. The loaded micelle of claim 10, wherein the hydrophobic block comprises a second carbonate repeat unit comprising no steroidal group.

15. The loaded micelle of claim 10, wherein the biologically active cargo material is a drug selected from the group consisting of paclitaxel, cyclosporin A, spironolactone, and combinations thereof.

16. A method, comprising:
forming a solution comprising a water miscible organic solvent, a biologically active cargo material, and an amphiphilic block copolymer, the block copolymer comprising a) a hydrophilic poly(alkylene oxide) block and b) a biodegradable hydrophobic block comprising a carbonate repeat unit of formula (3):





wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group; and
dialyzing the solution against water using a dialysis membrane system, thereby forming a loaded micelle comprising the block copolymer bound by non-covalent interactions to the biologically active cargo material.

17. The method of claim 16, wherein said dialyzing is performed at a temperature less than 10° C.

18. The method of claim 16, wherein the block copolymer and cargo material are present in the solution in a respective weight ratio of 15:1 w/w to 15:5 w/w.

19. A method of treating a cell comprising contacting the cell with the loaded micelle of claim 9.

20. A cyclic carbonate monomer having the formula (2):




wherein i) t and t′ are integers independently having a value from 0 to 6 wherein t′ and t cannot both be zero, ii) each Q1 is a monovalent radical independently selected from the group consisting of hydrogen, halides, alkyl groups comprising 1 to 30 carbons, and aryl groups comprising 6 to 30 carbon atoms, iii) L′ is a divalent linking group comprising one or more carbons, and iv) S′ is a steroidal group.

21. The monomer of claim 1, wherein t and t′ are each 1, each Q1 at carbon 4 is hydrogen, each Q1 at carbon 6 is hydrogen, and Q1 at carbon 5 is selected from the group consisting of hydrogen, methyl, and ethyl.

22. The monomer of claim 1, wherein S′ is a cholesteryl group: