Patent Application
20110144281
The present invention concerns polymers, and more particularly to amphiphilic polymers with a cholane core.
Compact structure and high segment density render star polymers with distinct physical properties in bulk, as a melt and in solution compared to their linear analogs. Star polymers with more than 3 arms, even up to 128 arms, have been studied from the synthetic and theoretical viewpoints (Inoue, K. Progress in Polymer Science 2000, 25, (4), 453-571; Hadjichristidis, N; Pitsikalis, M.; Pispas, S.; latrou, H. Chemical Reviews, 2001, 101, 3747-3792; Hirao, A.; Hayashi, M.; Tokuda, Y.; Haraguchi, N.; Higashihara, T.; Ryu, S. W. Polymer Journal 2002, 34, (9), 633-658; Vlassopoulos, D. Journal of Polymer Science Part B-Polymer Physics 2004, 42, (16), 2931-2941).
Poly(ethylene glycol) (PEG)-based star polymers have attracted much attention from researchers due to their well-known bioacceptability (Hawker, C. J.; Chu, F. K.; Pomery, P. J.; Hill, D. J. T. Macromolecules 1996, 29, (11), 3831-3838; Hou, S. J.; Taton, D.; Saule, M.; Logan, J.; Chaikof, E. L.; Gnanou, Y. Polymer 2003, 44, (18), 5067-5074; Lapienis, G.; Penczek, S. Macromolecules 2000, 33, (18), 6630-6632; Taton, D.; Saule, M.; Logan, J.; Duran, R.; Hou, S.; Chaikof, E. L.; Gnanou, Y. Journal of Polymer Science Part a-Polymer Chemistry 2003, 41, (11), 1669-1676). Branched PEG stars have shown to be more promising than linear PEG in certain biomedical and pharmaceutical applications. Much interest has been focused on asymmetric star polymers, where arms differ in molecular weight or chemical composition (Hirao, A.; Hayashi, M.; Tokuda, Y.; Haraguchi, N.; Higashihara, T.; Ryu, S. W. Polymer Journal 2002, 34, (9), 633-658; Beyer, F. L.; Gido, S. P.; Poulos, Y.; Avgeropoulos, A.; Hadjichristidis, N. Macromolecules 1997, 30, (8), 2373-2376; Tselikas, Y.; latrou, H.; Hadjichristidis, N.; Liang, K. S.; Mohanty, K.; Lohse, D. J. Journal of Chemical Physics 1996, 105, (6), 2456-2462). These star polymers are expected to exhibit interesting and unique physical performance owing to their branched asymmetrical architectures as well as hetero-phase structures.
Bile acids are surfactants biosynthesized in the liver of mammals as emulsifiers in the digestion of fats. Cholic acid, a major primary bile acid, possesses a rigid steroid skeleton structure and four hydrophilic groups located on one side of its rigid skeleton: three hydroxyl groups (all in α-position) and a carboxylic acid group. Several groups have synthesized polymers with either cholesterol cores or cholane cores, but due to solubility problems and incomplete polymerization, especially when all of the available OH groups are deprononated, the resultant polymers have been limited to a single hydrophilic chain (Kim et al. Langmuir, 2000, 16, 4792-4797; Han et al. Bioconjugate Chemistry, 2001, 12, 337-345; Koyama et al. Journal of Controlled Release, 2001, 77, 357-364). Thus, to date, amphiphillic polymers which have cholane cores, such as bile acids, in which all of the available derivatizable groups have been covalently bonded to hydrophilic polymer chains, have eluded synthesis.
We have discovered a novel class of amphiphilic polymers which have a cholane core structure with one or more hydrophilic polymer chains attached to the core. Furthermore, we have discovered that in an aprotic solvent, such as dimethylsulfoxide, the polymers can be produced by attaching the hydrophilic polymer chains by a “core first” method by partially deprotonating the cholane core before addition of the hydrophilic polymer chains. This significantly reduces or essentially eliminates the solubility problems that have prevented the successful synthesis of such polymers. In one example, a new polymer (CA-PEG4) has a core structure of cholic acid onto which four PEG chains are attached by the core-first and graft-from method. The cholane core imparts a spatial asymmetric distribution of the PEG chains, which are located on one side of the cholane backbone. Therefore, the polymer retains the hydrophobicity of cholic acid steroid skeleton on one face, while PEG chains modify its hydrophilicity on the other. Consequently, the amphiphilic asymmetric PEG stars can self-assemble into aggregates. Star polymers derived from cholic acid with poly(allyl glycidyl ether) arms have also been prepared similarly with well-defined molecular weight and low polydispersity. The double bonds on the polymer are used to introduce either amino groups or carboxylic acid groups to obtain amphiphilic polymers with cationic and anionic groups, respectively. The critical aggregation concentration in water changed with the arm length of the star polymer. A simple acetylation rendered the polymers thermosensitive, giving a cloud point ranging from 16 to 53° C. according to the degree of acetylation of the amino groups.
In one aspect of the present invention, there is provided a polymer comprising:
In another aspect of the present invention, there is provided a composition comprising:
In another aspect of the present invention, there is provided a micellar aggregate, the aggregate comprising:
In one aspect of the present invention, there is provided a polymer comprising:
In another aspect of the present invention, there is provided a composition comprising:
In another aspect of the present invention, there is provided a micellar aggregate, the aggregate comprising:
In another aspect of the present invention, there is provided an amphiphilic polymer, the polymer comprising:
In another aspect of the present invention, there is provided an amphiphilic polymer, the polymer comprising:
There is further provided in the present invention a polymer comprising:
The first monomer chain may comprise a large number of units, but preferably between 1 and 200 units.
The polymer of the present invention may further comprise a second monomer chain bonded to the first monomer chain, wherein the second monomer chain may optionally include a functional group adapted to be chemically modified.
The second monomer chain may comprise between 1 to 200 units.
Alternatively, the polymer of the present invention may further comprise a second monomer chain comprising at least one unit bonded on each unit of the first monomer chain, the at least one unit of the second monomer optionally including a functional group adapted to be chemically modified.
In the above polymer, the second functional group may be further modified, if necessary, to add additional monomer units. Also contemplated that the second functional group being selected from the group consisting of, but not limited to, transferrin, asialoglycoprotein, antibodies, antibody fragments, low density lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stem cell factors, erythropoietin, epidermal growth factor (EGF), insulin, asialoorosomucoid, mannose-6-phosphate, mannose, LewisX and sialyl LewisX, N-acetyllactosamine, galactose, lactose, thrombomodulin, fusogenic agents, polymixin B, hemagglutinin HA2, lysomotrophic agents, peptide, folic acid, and nucleus localization signals (NLS)
Typical functional groups include OH, NH2, SH, CO2H, amino acids, phosphates and the like and are located at the end of the chain and which may be further modified to attach peptides, proteins, nucleotides, glycopeptides, oligoglycerides, drug molecules, and the like.
In one example, the first and second monomer chain may form either block polymers or random copolymers. The derivatizable groups X covalently bonds the hydrophilic polymers to the cholane core. Typically, the derivatizable groups X include OH, NH2, SH, CO2H and the like, which when derivatized can form ethers, secondary or tertiary amines, thioethers, and esters or ketones, and the like.
Examples of the optional first functional group of one or both of the first and second monomer chain include, but not are not limited to, methyl, ethyl, halomethyl, haloethyl, allyl, vinyl, protected hydroxymethyl and hydroxyethyl, protected aminomethyl, aminoethyl, etc.), which themselves may be optionally further modified chemically; The functional groups on M or P units can be further extended to form branched polymers; M and P may form block or random copolymers;
The first monomer chain can be a compound selected from ethylene glycol or —CH2—CH2—O—, for example. The second monomer chain can be of the formula —CH2—CHR—O—, wherein R is H, CH3, CH2CH3, CH2Cl, CH2SH, CH2CH2SH, CH2OH, CH2CH2OH, CH2OCH2CH═CH2, for example.
In another aspect of the present invention, there is provided a process for preparing an amphiphilic polymer, the process comprising:
In another aspect of the present invention, there is provided a process for preparing CA-PEG4, the process comprising:
Further aspects and advantages of the present invention will become better understood with reference to the description in association with the following Figures, wherein:
Unless otherwise specified, the following definitions apply:
The singular forms “a”, “an” and “the” include corresponding plural references unless the context clearly dictates otherwise.
As used herein, the term “comprising” is intended to mean that the list of elements following the word “comprising” are required or mandatory but that other elements are optional and may or may not be present.
As used herein, the term “consisting of” is intended to mean including and limited to whatever follows the phrase “consisting of”. Thus the phrase “consisting of” indicates that the listed elements are required or mandatory and that no other elements may be present.
As used herein, the term “cholane” is intended to mean a class of steroid compounds which is characterized as having a hydrocarbon skeleton with four fused rings, generally arranged in a 6-6-6-5 members on the cycles. One example of a cholane includes, but is not limited to, cholic acid, which is a bile acid.
As used herein, the term “bile acid” is intended to mean a steroid structure with four fused rings, a five or eight carbon side chain terminating in a carboxylic acid group. Examples of bile acids include, but are not limited to, cholic acid, chenodeoxycholic acid, deoxycholic acid, lithocholic acid and their derivatives such as glycocholic acid and taurocholic acid.
As used herein, the term “hydrophilic polymer” is intended to mean repeating units of epoxy compounds based on an ethylene oxide structure. The polymer can have a plurality of units, preferably up to 1000 units and more preferably 80 units. Examples of such hydrophilic polymers include, but are not limited to, poly(ethylene glycol) (PEG) and poly(allyl glycidyl ether). One skilled in the art will readily recognize that many hydrophilic polymers are available and may be used to practice the present invention.
As used herein, the term “derivatizable group” is intended to mean a chemical functional group which may be reacted to another activated species to form a covalent bond between the species and the group. Examples of derivatizable groups include, but are not limited to, OH, SH, NH2, CO2H, and the like.
As used herein, the term “partially deprotonated” is intended to mean that at least one of several available derivatizable groups is deprotonated under conditions in which the deprotonated species remains soluble in the aprotic solvent used. Broadly speaking, between 5 and 99% deprotonation of the total number of derivatizable groups is desirable. In one example provided in the instant invention, partial depronation is 25% which means that one of the four OH groups of the cholic acid core was deprotonated to maintain solubility of the polymer. To ensure the success of the anionic polymerization: the deprotonated species should remain soluble in the aprotic solvent. In the example described herein, the aprotic solvent is DMSO. Furthermore, the degree of deprotonation has to be sufficient for the anionic polymerization to take place.
Broadly speaking, the present invention concerns amphiphilic polymers which have a cholane core having at least one derivatizable group covalently bonded to the core. The derivatizable groups can be OH, SH, NH2 or CO2H groups. In one example described herein, the derivatizable groups are OH groups. The invention also contemplates cores in which mixed derivatizable groups are covalently bonded to the fused ring system of the cholanes. Generally speaking, one or more derivatizable groups may be present. In one example described herein, four derivatizable groups are covalently bonded to the fused ring system of the cholane core. One or more of the derivatizable groups can be covalently bonded to a hydrophilic polymer chain. In the example described herein, four
PEG chains are covalently bonded to the respective deriavtizable groups. The chain length of the hydrophilic polymers are tunable to balance the amphiphilicity of the polymer.
Also contemplated by the present invention are compositions of the amphililic polymers. The compositions may further include carriers, fillers, excipients, and the like.
There is further provided in the present invention a polymer comprising:
a) a cholane core having between one and four derivatizable group covalently bonded thereto;
b) a first monomer chain bonded to the derivatizable group, wherein the first monomer chain may optionally include a first functional group adapted to be chemically modified; and
c) a second functional group located at the end of the first monomer chain.
The first monomer chain may comprise a large number of units, but preferably between 1 and 200 units.
The polymer of the present invention may further comprise a second monomer chain bonded to the first monomer chain, wherein the second monomer chain may optionally include a functional group adapted to be chemically modified.
The second monomer chain may comprise between 1 to 200 units.
Alternatively, the polymer of the present invention may further comprise a second monomer chain comprising at least one unit bonded on each unit of the first monomer chain, the at least one unit of the second monomer optionally including a functional group adapted to be chemically modified.
In the above polymer, the second functional group may be further modified, if necessary, to add additional monomer units. Also contemplated that the second functional group being selected from the group consisting of, but not limited to, transferrin, asialoglycoprotein, antibodies, antibody fragments, low density lipoproteins, interleukins, GM-CSF, G-CSF, M-CSF, stem cell factors, erythropoietin, epidermal growth factor (EGF), insulin, asialoorosomucoid, mannose-6-phosphate, mannose, LewisX and sialyl LewisX, N-acetyllactosamine, galactose, lactose, thrombomodulin, fusogenic agents, polymixin B, hemagglutinin HA2, lysomotrophic agents, peptide, folic acid, and nucleus localization signals (NLS)
Typical functional groups include OH, NH2, SH, CO2H, amino acids, phosphates and the like and are located at the end of the chain and which may be further modified to attach peptides, proteins, nucleotides, glycopeptides, oligoglycerides, drug molecules, and the like.
In one example, the first and second monomer chain may form either block polymers or random copolymers. The derivatizable groups X covalently bonds the hydrophilic polymers to the cholane core. Typically, the derivatizable groups X include OH, NH2, SH, CO2H and the like, which when derivatized can form ethers, secondary or tertiary amines, thioethers, and esters or ketones, and the like.
Examples of the optional first functional group of one or both of the first and second monomer chain include, but not are not limited to, methyl, ethyl, halomethyl, haloethyl, allyl, vinyl, protected hydroxymethyl and hydroxyethyl, protected aminomethyl, aminoethyl, etc.), which themselves may be optionally further modified chemically; The functional groups on M or P units can be further extended to form branched polymers; M and P may form block or random copolymers;
The first monomer chain can be a compound selected from ethylene glycol or —CH2—CH2—O—, for example. The second monomer chain can be of the formula —CH2—CHR—O—, wherein R is H, CH3, CH2CH3, CH2Cl, CH2SH, CH2CH2SH, CH2OH, CH2CH2OH, CH2OCH2CH═CH2, for example.
Specifically, the present invention concerns amphiphilic polymers, which may be asymmetric or may be so-called “star” polymers, and which are produced by the grafting of PEG chains of different lengths on the cholane core of cholic acid. We have achieved this by anionic polymerization of ethylene oxide, which provides polymers with very low polydispersity. It is easy to apply the same grafting method to other bile acids or compounds with multiple derivatizable functional groups of this kind. The PEGylated cholic acid derivatives can form spherical micellar aggregates in water, providing interesting reservoir for hydrophobic compounds that may be explored for use as drug delivery vehicles. The OH groups of the PEG chains may be further modified to introduce other functional groups for different applications. Further experiments will be carried out to study the formation of mixed micelles. Contemplated uses for the micelles or mixed micelles include, but are not limited to, drug carriers or cosmetics. Micelles can be prepared easily by dissolving the synthesized polymers in water above their critical micellar concentrations (CAC). Mixed micelles can be prepared by dissolving the amphiphilic polymers together with bile acids, fatty acids or other similar or different polymeric or oligomeric derivatives of bile acids. Active ingredient(s), particularly agents that are hydrophobic or amphiphilic can be incorporated into these micelles. The active ingredient can be release over time along with the disruption or solublization of the micelles.
Poly(ethylene glycol) (PEG) arms are grafted onto a cholane core via anionic polymerization to obtain asymmetric star-shaped polymers. The anionic polymerization of ethylene oxide was optimized in different solvents and with different degrees of deprotonation of the initiating hydroxyl groups on a cholic acid derivative. In dimethylsulfoxide, 25% deprotonation of the hydroxyl groups on the cholane core afforded a better control over the molar mass and polydispersity of the polymer obtained. Well-defined cholic acid-PEG stars (polydispersity index ca. 1.05) with tunable molar masses (ca. 1000-13000) were obtained and characterized by the use of size exclusion chromatography, MALDI-TOF mass spectrometry, NMR spectroscopy and thermal analysis. The critical aggregation concentrations of the star polymers were determined and spherical aggregates of the polymers with different PEG chain lengths were observed by transmission electron microscopy using the freeze-fracture etching technique and compared with results obtained from dynamic light scattering measurements.
A general method for the synthesis of the polymers of the present invention is shown below and is disclosed merely for the purpose of illustration and are not meant to be interpreted as limiting the processes to make the polymers by any other methods.
Cholic acid (98%) and 2-aminoethanol (98%) were purchased from Aldrich and used without further purification. Dimethylsulfoxide (DMSO, from Aldrich) was dried by refluxing with calcium hydride for 48 h before distillation. Tetrahydrofuran (THF) was dried with sodium in the presence of benzophenone and was distilled after the solution turned dark blue. Potassium naphthalene was prepared directly in dry THF from naphthalene (>99%, Aldrich) and potassium (98% in mineral oil, Aldrich) with a concentration of 0.45 mol/L (titrated with a standard hydrochloric acid solution). Ethylene oxide (EO) was distilled from a trap with a 1.6 mol/L n-butyl lithium solution in hexane (from Aldrich) to another trap after passing through a calcium hydride drying column. All glassware used in the anionic polymerization was flame-dried under vacuum before use.
Cholic acid methyl ester 2 (8.0 g), prepared from cholic acid via a previously published procedure (Benrebouh, A.; Zhang, Y. H.; Zhu, X. X. Macromolecular Rapid Communications 2000, 21, (10), 685-690), was dissolved in 50 mL of dry 2-aminoethanol and refluxed for 4 h. The reaction solution was then cooled and 50 mL of ice water was poured into the solution. The product was precipitated and filtered at room temperature, then dissolved in hot methanol followed by the addition of ethyl acetate (4 times excess) to precipitate again. After filtration and drying in an vacuum oven, 8.0 g of product (3) was obtained with a yield of 93%. Elemental analysis: C26H45NO6, calculated: C, 69.14%; H, 10.04%; N, 3.10%. found: C, 69.15%; H, 10.49%; N, 3.14%. FTIR (cm−1): 1655 (sharp), 3288 (sharp). 1H NMR (400 MHz, DMSO-d6, ppm): δ=0.58 (3H, s, 18-CH3), 0.81 (3H, s, 19-CH3), 0.92 (3H, d, J=6.4 Hz, 21-CH3), 1.0-2.2 (m, protons on the steroidal skeleton backbone), 3.08 (2H, q, J. 6.1 Hz, 25-NCH2), 3.18 (1H, m, 3-CH), 3.35 (2H, q, J. 5.7 Hz, 26-CH2O), 3.60 (1H, m, 7-CH), 3.77 (1H, m, 12-CH), 4.01 (1H, d, J=3.3 Hz, 12-COH), 4.10 (1H, d, J=3.5 Hz, 7-COH), 4.32 (1H, d, J=4.3 Hz, 3-COH), 4.62 (1H, t, J. 5.4 Hz, 26-COH), 7.75 (1H, t, J=5.7 Hz, 24-CONH). 13C NMR (100 MHz, DMSO-d6, ppm): δ=13.21, 17.99, 23.49, 23.67, 27.08, 28.16, 29.43, 31.26, 32.58, 33.36, 35.25, 35.26, 36.07, 36.18, 42.23, 42.26, 42.38, 46.59, 46.99, 60.84, 67.11, 71.30, 71.88, 173.65. LCMS: tR 10.36 min, m/z 452 M+H+.
Alternatively, the carboxylic acid group of bile acids can be reduced to the alcohol, among several methods, by the method used by Kihira et al. (Kihira, K.; Mikami, T.; Ikawa, S.; Okamoto, A.; Yoshii, M.; Miki, S.; Mosbach, E. H.; Hoshita, T. Steroids 1992, 57, 193-198). In a typical reaction, 10 g of CA (24.5 mmol) were dissolved in 400 mL of dry THF under nitrogen atmosphere with 13 mL (93.9 mmol) of triethylamine. To this solution, 8 mL (83.7 mmol) of ethylchloroformate were added dropwise and the solution left to react at room temperature for 2 hours. Then, 10.67 g of sodium borohydride dissolved in 11 mL of water were slowly added and reacted for 3 hours. After adding 200 mL of water and concentrated hydrochloric acid to neutralize the solution, the aqueous phase was extracted 3 times with ethyl acetate. The organic phase was dried over magnesium sulfate, filtered and evaporated. The solid residue was recrystallized in methanol and the resulting crystal dried in a vacuum oven. Yield: >96% of tetranol of CA.
In a typical procedure, all the glassware and needles were flame-dried under vacuum and purged with argon 3 times. 0.45 g of 3 (1 mmol) was placed into a 100 mL flask, charged with 40 mL of dry DMSO. The potassium naphthalene solution in THF (0.43 mol/L) was introduced (9.2 mL, 1 equiv., or 2.3 mL, 0.25 equiv.) dropwise into the vigorously stirred flask via a canula by high pressure argon. Then, dry ethylene oxide chilled in dry ice/acetone, was introduced into the flask and polymerized at 40° C. for 48 h. The reaction mixture was neutralized and quenched with concentrated HCl. The DMSO solution was extracted with hexane (50 mL×3) to remove naphthalene and DMSO was removed by distillation under vacuum. A small amount of THF was added to the residues to dissolve the polymer and to precipitate the salt. After filtering off the salt, the polymer was concentrated to dryness by rotary evaporation and high vacuum. The polymer was characterized by the use of various techniques as described below.
In a typical procedure, all glassware was flame-dried under vacuum and purge with argon three times. In a dry flask, 1 g of CA-OH24 (2.5 mmol) was placed in a 250 mL Schlenk flask and purged with argon. The flask was then charged with 67 mL of freshly distilled DMSO. A 6.3 mL (0.4 M, 2.5 mmol) of potassium naphthalene solution was added stepwise to the stirred solution of DMSO maintained at 30° C. To obtain 5 repeating-unit polymer chain length, 6.0 mL (5.78 g; 51 mmol) of freshly distilled allyl glycidyl ether (AGE) were transferred to the flask. The polymerization was let to proceed overnight and then quenched with concentrated hydrochloric acid and extracted with 3 portions (30 mL) of hexanes. Water (200 mL) was added to DMSO and extractions with dichloromethane (3×100 mL) were performed. The solvent was evaporated and the sample was dried under high vacuum. Yield: >90%.
Functional groups such as COOH or NH2 can be introduced as pendant groups of the polymer chains and examples of the procedures are described below:
The addition of carboxylic acid pendant groups was achieved by the addition of 3-mercaptopropionic acid on the CA-24OH(AGEn)4 was performed by dissolving first the polymer in THF in a ratio of 2.75 mL for 1 g of polymer. Then, 5 and 0.15 eq., according to the number of double bonds, of the 3-mercaptopropionic acid and AIBN were added, respectively, and the solution was refluxed for 5 hours. The most volatile compounds were removed with a rotary evaporator and a vacuum distillation was performed to remove the less volatile ones. The viscous liquid obtained from this distillation was purified by dialysis.
Yield: >94%.
The addition of amino Pendant Groups was carried out as the following: CA-24OH(AGEx)4 was dissolved in methanol ( 1/20, w/v), 5 equivalents of cysteamine hydrochloride and 0.5 equivalent of AIBN per double bond were added. The solution was refluxed for 36 hours. Methanol was evaporated and the residual syrup was dissolved in chloroform. Sodium hydroxide pellets were added to neutralize the ammonium chloride salt and the organic layer was washed with water. After solvent evaporation, the resulting viscous liquid was dialyzed against deionized milli-Q water. Yield>95%.
The infrared spectra were recorded on the Bomen MB-100 Fourier transform IR spectrometer at room temperature, potassium bromide pellets were made with ground polymer. 1H NMR spectra of polymers and cholic acid derivatives were recorded on a Bruker AMX400 spectrometer operating at 400 MHz for protons. The molar mass of the polymers were determined by size exclusion chromatography (SEC) in THF at 25° C. with a flow rate of 1 mL/min in reference to linear PEG standards. The thermal transitions of the polymers were analyzed on a TA DSC2910 differential scanning calorimeter (DSC).
MALDI-TOF mass spectrometry was performed on a Bruker Autoflex MALDI-TOF mass spectrometer, which used a 20 kV extraction voltage and a N2 laser of 337 nm wavelength. Dithranol (1,8-dihydroxy-910H-anthralenone) (Sigma) was used as a matrix with the addition of LiCl for the MALDI-TOF MS analysis. A peptide calibration standard with a molecular weight range of 1-4 kDa and a protein calibration standard with a molecular weight range of 3-25 kDa were used to calibrate the molar masses of the star polymers.
Liquid chromatography ultra-violet conditions: Betasil C18 column 150×4.6 mm, A=5% v/v trifluoroacetic acid (TFA) in H2O and B=5% v/v TFA in CH3CN, gradient of B from 20% to 80% over 20 min, flow rate at 0.5 mL/min., injection volume: 10 μL. MS conditions: scan 100-800, cone voltage 30 kV, temperature 400° C., mode (polarity) positive. Surface tension (ST) measurements were performed on a First Ten Angstroms instrument model FTÅ200 with milli-Q water. The pendant droplet method was used to calculate the critical aggregation concentration (CAC) of the polymers. The instrument was calibrated using the needle width as reference.
The average size of the aggregates was measured by dynamic light scattering (DLS) on a Brookhaven Instrument (model BI-200SM) equipped with a 532 nm laser. For the size distribution of the different samples in solution, the inverse Laplace transform was performed with MatLab using the regularization function. The average hydrodynamic radius (Rh) was calculated according to the apparent F of the Williams-Watts function.
For transmission electron microscopy (TEM), different sample concentrations were prepared in milli-Q water for freeze-fracture. A small amount of these solutions were dropped onto a good sample carrier, and then frozen in liquid propane. The frozen samples were then mounted on the sample holder of a BAL-TEC freeze etching instrument (model BAF060). Samples were then fractured, let sublimate for less than 30 seconds before the newly created surface was coated from an angle of 45° (shadowed) with 2 nm of platinum-doped carbon. A 10 nm layer of carbon was then applied perpendicularly. The samples were placed in distilled water to make the platinum-carbon replica float on the water surface. They were then deposited on carbon-coated copper grids. The replicas were examined on a JEOL JEM-2000FX TEM operating at an acceleration voltage of 80 kV.
Cholic acid methyl ester was aminolyzed with 2-aminoethanol (Scheme 1). The FTIR spectrum of 3 shows very sharp peaks at 1655 cm−1 (C=0), 3288 cm−1 (NH) and 1570 cm−1 (NH) typical of an amide and no peaks attributable to an ester (1736 cm−1 for 2). This indicates the complete conversion of the methyl ester reacts to the corresponding amide.
1H NMR and 13C NMR spectra also confirm the structure of the amide 3.
After deprotonation with potassium naphthalene, the solubility of the alkoxide becomes even lower due to the tendency of the alkoxide to aggregate; the reaction solution turned milky and opaque. DMSO was used as a solvent to increase the solubility of the salted precursor 3 in polymerization. It was observed that the phase separation occurred when the monomer was introduced less than 40 equiv. in order to generate short PEG chains, and polymers with wide PDI were obtained. A homogenous polymer solution was obtained when 80 equiv. of EO was charged into the reaction mixture and a narrow dispersed polymer was isolated (PDI: 1.07), even though the mixture still became milky at the beginning stage of polymerization with 100% deprotonation of precursor 3.
Although narrow disperse polymers can be obtained using a full deprotonation of the hydroxyl groups with large quantities of monomer added (usually more than 80 equiv.), phase separation are still encountered in the preparation of star polymers with short PEG chains. Star PEG polymers can be prepared with a partial deprotonation of alcohol groups due to the rapid proton exchange between the dormant hydroxyl groups and the active alkoxides. The proton NMR study of the prepared star-shaped polymer showed that four PEG chains were attached on one cholane core.
In order to avoid the milky transition, 25% deprotonation (Scheme 1) was employed in the in the preparation of samples I to VI. The polymerization solutions remained transparent throughout the entire duration of the polymerization. As shown in Table 1, a series of star polymers were prepared with narrow polydispersities (
FTIR spectra show a decrease in the carbonyl band at 1641 cm−1 with the increasing length of PEG chains grafted on the cholane core 3 from sample I to sample V (data not shown), indicating a qualitative chain growth. It is important, however, to ensure that all four hydroxyl groups on the cholane core are grafted with a PEG chain when 25% deprotonation of the hydroxyl groups were used. To do this, the CA-PEG4 star polymers can be treated with the trifluoroacetic anhydride, followed by NMR analysis of the integral of the proton signal intensity of the CH2 adjacent to the trifluoroacetyl group.
The theoretical values of molecular weight of the star polymers can be calculated based on the amounts of initiator and monomer (EO) added. However, SEC and MALDI-TOF MS analyses gave lower molecular weights than the theoretical values (Table 1). It is known the star-shaped polymers have a smaller hydrodynamic radius than the corresponding linear polymers with the same molecular weight. Therefore, when linear PEGs were used as the calibration standards, the molecular weights of star-shaped PEGs may be under-estimated in SEC analysis.
Both MALDI-TOF MS and NMR can be used for the accurate measurements of polymers of lower molar masses. The molar masses calculated from the 1H NMR signals, using the ratio of the proton signals of PEG chains and the methyl group (position 18) on cholic acid, are closer to the theoretical molecular weights of the CA-PEG4 stars than the SEC results. MALDI-TOF MS is particularly suitable in the analysis of polymers of low polydispersity of molar masses. For the CA-PEG4 stars with lower molar masses (samples I to IV), high resolution spectra were obtained by MALDI-TOF (
Melting point suppression is a well-known effect of the PEG chains in star polymers, because of the defective PEG crystal lattice caused by the core and by the lower molecular weight of the PEG chains (Chen, E. Q.; Lee, S. W.; Zhang, A.; Moon, B. S.; Honigfort, P. S.; Mann, I.; Lin, H. M.; Harris, F. W.; Cheng, S. Z. D.; Hsiao, B. S.; Yeh, F. Polymer 1999, 40, (16), 4543-4551; Chen, E. Q.; Lee, S. W.; Zhang, A. Q.; Moon, B. S.; Mann, I.; Harris, F. W.; Cheng, S. Z. D.; Hsiao, B. S.; Yeh, F. J.; von Merrewell, E.; Grubb, D. T. Macromolecules 1999, 32, (15), 4784-4793). The melting points of crystalline polymers may also depend on the thermal history of the sample. In order to erase the thermal history of the samples, the DSC thermograms of the CA-PEG4 samples were recorded during the second heating at 10° C./min (
Because of the asymmetric structure and the amphiphilicity of the CA-PEG4 star polymers, they easily aggregate in water. The aggregation of these polymers was then studied by the use of surface tension technique. Molal concentration (mol of solute per 1 kg of the solvent) was used because of its convenience in the calculation of concentration for samples of varying volume but of known weight. The value should be close to the molar concentration (mol of solute per liter of solution) at low concentrations. The surface tension of the solution should decrease with increasing concentration of surfactant molecules and becomes stable above a certain concentration of the surfactants, which can be defined as the CAC of the surfactant. While the hydrophobic parts of the surfactants are unchanged, the decrease in ST with the increasing hydrophilic segments in the surfactant is almost invariable.
TEM images of the star polymers shown in
TEM provided images of a limited number of the frozen micelles (not large enough to provide a statistical distribution of the size). DLS experiments can be used to study the size and distribution of the micelles in solution. Selected samples were studied and Rh is calculated according to the Stoke-Einstein equation assuming a spherical structure, which is not the case for the aggregates of sodium cholate, leading to a smaller hydrodynamic diameter than the average rod length. The large width of the distribution as shown by the β value can be explained by the stepwise aggregation of sodium cholate that unavoidably gives many species in solution. For samples I and III, the average hydrodynamic diameters are larger than those shown in the TEM images. The discrepancy is not too large and could be due to the more hydrated state of the micelles in solution.
Cytotoxicity Test: The MTT assay and the MTS assay are laboratory tests and standard colorimetric assays for measuring the activity of enzymes that reduce MTT or MTS+PMS to formazan, giving a purple color. This mostly happens in mitochondria, and so the assays are therefore largely a measure of mitochondrial activity. It can be used to determine the cytotoxicity of materials. Agents with cell toxicity result in mitochondrial dysfunction. Yellow MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole) is reduced to purple formazan in the mitochondria of living cells. The absorbance of this colored solution is quantified by measuring at a wavelength between 500 and 600 nm on a spectrophotometer. The standard MTT assays were carried out with three CA-PEG4 polymers (1850<Mn<15000). Cell viability remained 100% within experimental error for a concentration of the CA-PEG4 polymers up to 0.1 mg/ml and 80% for a concentration of the CA-PEG4 polymers at 10 mg/ml, indicating very low cytotoxicity of the polymers.
Loading of a drug. Ibuprofen has a very limited solubility in neutral water and remains as a crystalline residue when added in water, even after vigorous agitation. The addition of CA-PEG4 (Mn=1850 and 2400) to the system increased the miscibility of the water-ibuprofen mixture. The gradual addition of CA-PEG4 (above the expected CMC at ca. 15 mM) produced a cloudy emulsion and then a clearer solution, indicating the solubilisation of the drug ibuprofen in the micellar system.
5β-Cholane-3α,7α,12α,24-tetrol (CA-OH) was prepared from cholic acid according to a literature procedure (K. Kihira, T. Mikami, S. Ikawa, A. Okamoto, M. Yoshii, S. Miki, E. H. Mosbach and T. Hoshita, Steroids, 1992, 57, 193-198). We used anionic polymerization where the alcoholate groups of CA-OH form ether linkages upon ring opening of the oxirane derivatives. Allyl glycidyl ether (AGE) is used to allow the subsequent addition of thiolated compounds containing carboxylate or amino groups. Naphthalene radical anions were obtained by mixing of naphthalene and potassium in anhydrous tetrahydrofuran at an approximate concentration of 0.40 M and further titrated with a standard 0.1 M hydrochloric acid aqueous solution. AGE (≧99%) and dimethyl sulfoxide (DMSO, ≧99%) were obtained from Aldrich and dried over calcium hydride prior to reflux (2 hours) under reduced pressure followed by distillation.
In a typical procedure, all glassware was flame-dried under vacuum and purged with argon three times. In a dry flask, 1 g of CA-OH (2.5 mmol) was placed in a 250 mL Schlenk flask and purged with argon. The flask was then charged with 67 mL of freshly distilled DMSO with the aid of a double-ended needle using a positive pressure. Potassium naphthalene solution (6.3 mL, 2.5 mmol) was added stepwise, and after the disappearance of the green color, slowly to the stirred solution of DMSO maintained at 30° C. In order to obtain 5 repeat units in length for every initiating site on CA-OH, 6.0 mL (5.78 g, 51 mmol) of freshly distilled AGE were transferred to the flask in the same manner as DMSO. The polymerization was allowed to proceed overnight and finally quenched with concentrated hydrochloric acid and extracted with hexanes (3×30 mL). Water (200 mL) was added to the DMSO medium and extractions with dichloromethane (3×100 mL) were performed, followed by the removal of the solvent on a rotary evaporator. Final traces of solvent were removed under high vacuum. Yield: >90%. 1H NMR (400 MHz, CDCl3, ppm, n=5): spectrum shown in
The addition of 3-mercaptopropionic acid on the CA-OH(AGEn)4 was performed by dissolving 1 g of the polymer in 2.75 mL THF. Then, 5 and 0.15 equivalents, according to the number of double bonds, of 3-mercaptopropionic acid and AIBN were added successively and the solution was refluxed for 5 hours. The most volatile compounds were removed with a rotary evaporator and a vacuum distillation was performed to remove the less volatile ones. The viscous liquid resulted from this distillation was purified by dialysis. Yield: >94%, efficiency of the subsequent addition of thiolated compounds: 85% to 95%, see Table 2. 1H NMR (400 MHz, acetone-d6); 0.74 (18-CH3), 1.87 (4H, protons on the β carbon to the oxygen atom on the lateral chain and those on the carbon adjacent to the carboxyl group), 2.65 and 2.80 (4H, protons on the two carbons adjacent to the sulfur atom), 3.59 (5H, protons on the polymer backbone and those on the methylene groups connecting the lateral chains to the polymer backbone). 13C NMR (400 MHz, DMSO-d6, ppm, for n=5): 27.31, 28.68, 30.24, 35.47, 52.28, 69.70, 79.04, 173.93.
CA-OH(AGEn)4 was dissolved in methanol ( 1/20, w/v), 5 equivalents of cysteamine hydrochloride and 0.5 equivalent of AIBN per double bond were added. After refluxing the solution for 36 hours, methanol was evaporated and the residual syrup was dissolved in chloroform. Sodium hydroxide pellets were added to neutralize the ammonium chloride salt and the organic layer was washed one time with water. After solvent evaporation, the resulting viscous liquid was purified by dialysis. Yield: >95%, efficiency of the subsequent addition of thiolated compounds: 80% to 102%, see Table 2. 1H NMR (400 MHz, CDCl3, n=5): 0.70 (18-CH3), 1.86 (protons on the β carbon to the oxygen atom on the lateral chains), 2.08 (protons on the nitrogen atom), 2.63 (4H, protons on the two carbons adjacent to the sulfur atom), 2.90 (2H, protons on the carbon adjacent to the amino group), 3.55 (5H, protons on the polymer backbone and those on the methylene groups connecting the lateral chains to the polymer backbone). 13C NMR (400 MHz, methanol-d4, ppm, for n=5): 23.43, 27.75, 28.24, 29.95, 34.50, 40.70, 69.91, 71.12, 79.19.
CA-OH(AGE5-NH2)4 was dissolved in dry dimethylformamide (1:2 w/v) and freshly distilled acetic anhydride was added dropwise. After 45 minutes, solid sodium hydroxide was added to the solution to neutralize the acidic medium. The solution was dialyzed against water and freeze-dried. Yield: 75% 1H NMR (400 MHz, CDCl3, n=5 with 5% acetylation): 0.68 (18-CH3), 1.87 (protons on the β carbon to the oxygen atom on the lateral chains), 2.01 (CH3-amide), 2.22 (protons on the nitrogen atom), 2.62 (4H, protons on the two carbons adjacent to the sulfur atom), 2.88 (2H, protons on the carbon adjacent to the amino group), 3.53 (5H, protons on the polymer backbone and those on the methylene groups connecting the lateral chains to the polymer backbone). 13C NMR (400 MHz, CDCl3, ppm, for n=5 with 5% acetylation): 23.61, 28.81, 30.10, 36.54, 39.20, 41.44, 51.17, 70.14, 71.30, 79.17, 171.03.
In order to find the degree of thiolation onto the allylic functions, back titrations were performed. The sample (0.1 g) was first dissolved in 10 mL of 0.1 M standardized solution of HCl or NaOH for the amine and acid series, respectively, and then 20 mL of milli-Q water were added. The back titration with acid, for CA-OH(AGE5-COOH)4, or base, for CA-OH(AGE5-NH2)4, was started and the pH was monitored in order to calculate the pKa, the first and the second equivalent points (Table 2).
Table 2 presents the molecular weight and polydispersity obtained by 1H NMR and SEC for the allylic CA-OH(AGEn)4 polymers. The 1H NMR results are in good accordance with the experimental feed ratios.
1H NMR was used to confirm the star-shaped architecture of the polymers. The alcohol end groups were reacted with trifluoroacetic anhydride which shifted the adjacent protons to a different chemical shift (5.36 ppm,). The ratio of the integrations of these protons to the double bond protons yields the polymer arm length. The results indicate that all four positions (3, 7, 12, 24) have been initiated.
We used 1H NMR, acid-base titration and elemental analysis to verify the completion of the addition of the thiol groups onto the double bonds. Both 1H NMR and elemental analysis provided similar values. In the case of the titration, data obtained still agree with the two previous methods for the amines CA-OH(AGEn-NH2)4, but not for the acids CA-OH(AGEa-COOH)4. The lower degree of substitution determined by titration reflects the difficulty to perform a complete titration.
These star polymers were designed to modify the aggregation properties of cholic acid, which aggregates around 8 mM when the carboxylic group is ionized (pKa 4.6 to 5.0) (S. Reis, C. G. Moutinho, C. Matos, B. de Castro, P. Gameiro and J. Lima, Anal. Biochem., 2004, 334, 117-126). The attachment of amino and carboxylic functions on cholic acid should change the range of pH where aggregation occurs and also the critical aggregation concentration (CAC) depending on the polymer chain length on the steroidal backbone.
Conductivity is a reliable and sensitive method to determine the critical micellar concentration (CMC). Conductivity experiments were performed with an Orion conductivity cell 018010 in a thermostated bath. The samples were prepared with standardized sodium hydroxide or hydrochloric acid solutions in milli-Q water to obtain the desired final concentration. The conductivity of the solution was measured after equilibration. The solution was then diluted to the next concentration with milli-Q water and the measurements were repeated. The results in Table 2 show that the CAC shift to a lower concentration as the polymer chain length increases due to the lipophilic nature of the uncharged polymeric units.
A simple addition of acetic anhydride to the amine set CA-OH(AGEa-NH2)4 results in the formation of the amide bonds on the amino groups to produce a statistical copolymer (see
Cloud points (CP) were measured with a BioCary 300 UV-Vis spectrometer at 500 nm with solutions of the polymer in milli-Q water (0.1 wt %). Samples were successively heated and cooled at a rate of 0.1° C./min. CPs were taken as the onset of the heating cycles. Polymers with different degrees of acetylation, as evidenced by 1H NMR, show different CPs. It is thus possible to tune the thermosensitivity by simply controlling the amount of acetic anhydride added to the polymers. The acetylation reaction is a simple reaction allowing an easy tuning of the CP. In this study, the polymers tested have CPs ranged from 16 to 53° C. (
1H
aError estimated to 7% for 1H NMR
bInitial average number of double bonds determined by 1H NMR
cThe first value is calculated according to the first equivalent point and the second is calculated according to the volume difference between the two equivalent points
dBased on the sulfur content
eMeasured at 25° C. and with 60% NaOH added for the acid series CA-OH(AGEn-COOH)4 or 10% HCl added for the amine series CA-OH(AGEn-NH2)4
From the foregoing description, it will be apparent to one of ordinary skill in the art that variations and modifications may be made to the invention described herein to adapt it to various usages and conditions. Such embodiments are also within the scope of the present invention.
All literature, patents, published patent applications cited herein are hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/CA2008/002151 | 12/5/2008 | WO | 00 | 3/2/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 60992543 | Dec 2007 | US |
