Article pubs.acs.org/Macromolecules
Glucose-Functionalized, Serum-Stable Polymeric Micelles from the Combination of Anionic and RAFT Polymerizations Ligeng Yin,† Molly C. Dalsin,† Antons Sizovs,‡ Theresa M. Reineke,*,† and Marc A. Hillmyer*,† †
Department of Chemistry, University of Minnesota, 207 Pleasant St. SE, Minneapolis, Minnesota 55455-0431, United States Department of Chemistry, Virginia Tech, Blacksburg, Virginia 24061, United States
‡
S Supporting Information *
ABSTRACT: Poly(ethylene-alt-propylene)−poly[(N,N-dimethylacrylamide)-grad-(2-methacrylamido glucopyranose)] (PEP−poly(DMA-grad-MAG), or PG) diblock terpolymers were synthesized by combining anionic and reversible addition−fragmentation chain transfer (RAFT) polymerizations. An ω-trithiocarbonate-functionalized PEP homopolymer served as the macromolecular chain transfer agent (macroCTA), and RAFT copolymerizations of DMA and a trimethylsilyl-protected MAG (TMS-MAG) monomer gave a family of PG diblock terpolymers after hydrolysis. The terpolymers had similar degrees of polymerization, and the MAG content ranged from 3.5 to 39 mol % in the hydrophilic block. At 70 °C, the reactivity ratios of DMA (1) and TMS-MAG (2) were determined to be r1 = 1.86 ± 0.07 and r2 = 0.16 ± 0.01, and thus the poly(meth)acrylamide blocks in the PG diblock terpolymers were likely to be gradient copolymers. Micellar dispersions from PG diblock polymers in water were examined by cryogenic transmission electron microscopy (cryo-TEM) and dynamic light scattering (DLS). Spherical micelles with core radii of ca. 7 nm and overall hydrodynamic radii of ca. 15 nm were the predominant morphologies observed in all samples prepared by sequential nanoprecipitation and dialysis. The electron-dense MAG moieties greatly increased the native contrast of the micellar coronae, which were clearly viewed as gray halos around the micellar cores in samples with relatively large MAG content. The stability of the glucose-installed micelles was tested in four biologically relevant media, from simple phosphate-buffered saline (PBS) to fetal bovine serum (FBS), using a combination of DLS and cryo-TEM measurements. Micellar dispersions from a PG diblock terpolymer with 16 mol % of MAG of the hydrophilic block were stable in 100% FBS over at least 14 h, suggesting their minimal interactions with serum proteins. Control experiments suggested that micelles composed of PDMA alone in the corona had similar serum stabilities. These sugarfunctionalized micelles hold promise as in vivo drug delivery vehicles to possibly prolong circulation time after intravenous administration.
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INTRODUCTION Polymeric micelles, which can physically encapsulate or chemically conjugate molecules of interest in their hydrophobic cores, hold great potential for the controlled release of drugs after intravenous injection. The advantages of these nanoscopic carriers include improved loadings of hydrophobic drugs over the drugs themselves in aqueous biological media, prolonged circulation time, and passive targeting of malignant tissues based on the enhanced permeation and retention (EPR) effect.1 Micelles are formed by the spontaneous aggregation of amphiphilic molecules in water, in which the hydrophobic components form the dense cores and the hydrophilic units reside in the micelle coronae. Ideal hydrophilic polymeric coronae should (i) shield and solublize the hydrophobic cores and their contents to increase the dose and bioavailability of a low- or nonsoluble drug, (ii) have minimal nonspecific interactions with blood components to avoid opsonization and minimize uptake by the mononuclear phagocyte system,2 and (iii) target malignant cells via both passive (via particle size) and active targeting methods with high specificity (e.g., © XXXX American Chemical Society
through site-specific ligands) while producing minimal side effects on healthy cells.3 To date, poly(ethylene oxide) (PEO) has been the most widely used hydrophilic polymer in polymeric micelles due to its minimal interactions with blood proteins, diminished enzymatic degradation, wide solubility in both organic solvents and water, and ready commercial availability. 4 However, PEO is not ideal, and various disadvantages in practice have been gaining attention, including thermal instability, hypersensitivity after intravenous and oral administrations,4 and accelerated blood clearance phenomenon due to the production of anti-PEO antibodies.5 Moreover, functionalizing PEO for targeted delivery is limited as there is typically only one reactive site at the end of the PEO chain.6 Also, the flexible PEO chains can result in the functionalized ends being buried within the PEO corona, thus decrease Received: January 30, 2012 Revised: April 13, 2012
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dx.doi.org/10.1021/ma300218n | Macromolecules XXXX, XXX, XXX−XXX
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Scheme 1. Synthesis of α-2-Deoxy-2-methacrylamido-1,3,4,6-tetra(O-trimethylsilyl)-D-glucopyranose (TMS-MAG, 2)
resulting diblock copolymers self-assembled into micelles with glucose-functionalized coronae, and both spherical and rodlike micelles in a dry, collapsed state were imaged by transmission electron microscopy (TEM). Similarly, thermoresponsive or pH-responsive micelles with glucose or mannose installed in the coronae have been obtained by covalently connecting the corresponding glycopolymers with polymers that exhibit LCST behavior in water such as poly(N-isopropylacrylamide)13 and poly(di(ethylene glycol) methyl ether methacrylate)17a or pHsensitive polymers such as poly(2-(diethylamino)ethyl methacrylate).12g Micelles with glycopolymer shells were also obtained from more complicated ABA,12c,d BAB,12e star-shaped (BA)4,12f and miktoarmed A3B block copolymers.12i Besides PMAU11b and poly(acrylate)s12h,17b that are slightly hydrophobic, more nonpolar polymers such as polystyrene12a and poly(ε-caprolactone)12e,f,i have also been used, which are potentially desirable in solubilizing more hydrophobic drugs based on solubility considerations.20 In these cases, the glycomonomers are usually protected with nonpolar groups (e.g., acetyl12a and isopropylidene12i) to afford a homogeneous mixture during polymerization, since glycopolymers are generally only soluble in very polar solvents such as water or water/alcohol mixtures. In this report, we have investigated glucose-functionalized diblock terpolymers consisting of aliphatic poly(ethylene-altpropylene) (PEP) as the hydrophobic component and a glucose-functionalized11b block as the hydrophilic component. This polymer series consist of blocks with highly disparate solubility properties. PEP is a macromolecular saturated hydrocarbon with a low glass transition temperature (Tg) (ca. −65 °C) and has been used in our structural design to eventually encapsulate drug candidates with very low water solubility (e.g., ellipticine21). A poly(meth)acrylamide hydrophilic block is being examined here for its ability to perform as a PEO alternative to mediate micelle aggregation in the presence of physiological salt and serum. We developed an efficient method for the synthesis of a trimethylsilyl (TMS)-protected monomer, α-2-deoxy-2-methacrylamido-1,3,4,6-tetra(O-trimethylsilyl)-D-glucopyranose (TMS-MAG) for the RAFT polymerizations. This monomer was copolymerized off the PEP macro chain transfer agent with N,N-dimethylacrylamide (DMA) to generate a family of PEP−poly(DMA-grad-MAG) (PG) diblock terpolymers with MAG mol % of the hydrophilic blocks ranging from 3.5 to 39 mol %. We prepared the micellar dispersions of PG diblock terpolymers in water via direct dissolution or nanoprecipitation and examined the selfassembled nanostructures by a combination of dynamic light scattering (DLS) and cryogenic transmission electron microscopy (cryo-TEM). Finally, we studied the colloidal stability of the glucose-functionalized micelles in four different biologically relevant media, from simple phosphate-buffered saline (PBS) to full fetal bovine serum (100% FBS), using DLS and cryo-TEM. PEP−PDMA (PA) and PEP−PEO (PO) diblock copolymers
targeting efficiency. Therefore, there is a need to explore possible PEO alternatives with enhanced functionality.7 Glycopolymers are synthetic macromolecules with carbohydrate moieties in the backbone or as pendant groups.8 Natural carbohydrate-based materials have been widely used in food, pharmaceutical, and medical applications due to their biocompatibility. Moreover, carbohydrates at the cell surface are recognized to undergo interactions with proteins and other biological entities (e.g., cells, pathogens) and thus play an important role in numerous cellular recognition processes, including cell adhesion, cellular trafficking, cancer cell metastasis, and immune response.9 Glycopolymers have various advantages over PEO as the stealth layers of micellar carriers, including tunable hydrophilicity and more available hydroxyls for functionalization. Moreover, the chemical structures and dispersity of synthetic carbohydrates can be precisely controlled to systematically understand the structure−function relationship.10 Therefore, incorporation of carbohydrate moieties into polymeric micelles holds potential for improved functionality and thus targeting efficiency and specificity. To date, many synthetic efforts on glycopolymers have been undertaken. For example, glycomonomers with olefinic groups have been synthesized, including (meth)acrylamides,11 (meth)acrylates,12 vinyltriazole,13 styrene derivatives,14 vinyl ethers,15 and norbornene derivatives.16 Monosaccharides such as glucose,11,12e−h,15,16 galactose12a,i and mannose,12b,13 and disaccharides such as lactose12c,d,14 have all been incorporated. Glycopolymers with controlled architecture and molecular weight have been prepared by numerous controlled polymerization techniques such as controlled radical11−15 and ringopening metathesis16 polymerizations as well as postpolymerization modifications.17 Reversible addition−fragmentation chain transfer (RAFT) polymerization is particularly versatile due to its good control of (meth)acrylamides, relatively fast polymerization rates, vast tolerance of solvents, and no metal catalyst needed.11,12b,g,13,15 Furthermore, thiocarbonate moieties can be quantitatively removed via facile aminolysis18 to eliminate the potential biological implications of this moiety and provide unique chemical handles for functionalizing with targeting moieties or imaging agents. A small number of studies have been published on glycopolymers in polymeric micelles, though many studies on sugar-based surfactants are in the literature.19 In the simplest case, a hydrophobic polymer is covalently connected to a glycopolymer, the latter of which can be chosen to be strongly hydrophilic, to form an AB-type amphiphilic diblock copolymer, in which A and B denote the hydrophobic and hydrophilic component, respectively. For example, Stenzel and co-workers developed a hydrophilic, glucose-functionalized monomer, 2methacrylamido glucopyranose (MAG), and copolymerized it using RAFT with a relatively hydrophobic, uridine-functionalized monomer, 5′-O-methacryloyl uridine (MAU), to give amphiphilic PMAU−PMAG diblock copolymers.11b The B
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Scheme 2. Synthesis of PEP−Poly(DMA-grad-MAG) Diblock Terpolymers
determined by DSC at a heating rate of 10 °C min−1. PEP-OH was esterified with a trithiocarbonate chain transfer agent (CTA)24 to afford the macromolecular CTA, PEP-CTA (3). DMA and TMS-MAG were copolymerized in the presence of PEP-CTA to afford the glucose-functionalized diblock terpolymers, as shown in Scheme 2. The composition of the two monomers in the feed was adjusted from [DMA]0:[TMSMAG]0 = 1:1 to 22:1 (Table S2). The values in the parentheses of the sample IDs indicate the Mn of the two blocks in kg mol−1, followed by the mole fractions of TMS-MAG in the hydrophilic blocks, as determined by 1H NMR spectroscopy. In all cases, the conversions of DMA were larger than those of TMS-MAG. The reactivity ratios of DMA (as monomer 1) and TMS-MAG (2, as monomer 2) were determined to be r1 = 1.86 and r2 = 0.16 in free-radical polymerizations at 70 °C (vide inf ra). On the basis of this, we infer that the poly(meth)acrylamide copolymer blocks were gradient in nature.25 SEC characterizations of a set of TMS-protected PG samples indicated that they were monomodal with apparent D̵ values in the range between 1.18 and 1.31 (Figure S6 and Table S2). The TMS groups were removed through acid-catalyzed deprotections with methanol. The deprotection was quantitative and nearly instantaneous; 2 min of reaction at 25 °C led to >99% removal of the TMS groups as determined by 1H NMR spectroscopy.26 An example is shown in Figure S5 for PG(3− 19−0.08). As the last step, we removed the trithiocarbonate CTA fragment through mild aminolysis with n-butylamine followed by reaction with an acrylate.18 Quantitative removal was evidenced by the disappearance of the characteristic absorbance peak of the trithiocarbonyl moieties at 309 nm in UV−vis spectroscopy (Figure S7). This step eliminated not only the possibility of aggregation due to the relatively long, hydrophobic C12 groups during micellization27 but also any potential toxicity from the trithiocarbonyl CTA fragments.28,29 Two samples, PA(3-21) and PO(3-25) with similar compositions to the PG diblock terpolymers but did not contain any glucose moieties in the hydrophilic block, were also prepared. Both samples were made from the same batch of PEP-OH23 as those in PG diblock terpolymers. PA(3-21) was made by the RAFT polymerization of DMA in toluene at 70
with similar overall molecular weight and compositions served as diblock copolymer controls that lack the polymerized glucose moieties.
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RESULTS AND DISCUSSION
Synthesis of α-2-Deoxy-2-methacrylamido-1,3,4,6tetra(O-trimethylsilyl)-D-glucopyranose (TMS-MAG, 2). The trimethylsilyl-protected 2-deoxy-2-methacrylamido glucopyranose monomer, TMS-MAG (2), was synthesized following a two-step procedure, as shown in Scheme 1. Glucosamine was first trimethylsilylated with N,O-bis(trimethylsilyl)acetamide in pyridine, and then the TMS-protected glucosamine (1) was reacted with methacryloyl chloride in DMF. The nonpolar nature of trimethylsilyl-protected 2 allowed purifications via simple extractions with hexanes, which yielded pure products from both steps. It should be noted that we obtained only the α-anomer based on the chemical shifts of H1 (doublet, 5.06 ppm, J12 = 3.55 Hz) and Ca (92.69 ppm) as shown in the 1H and 13C NMR spectra of TMS-MAG in Figures S1 and S2, which was consistent with a previous report. 22 The trimethylsilyloxy groups adjacent to Ca were on the same side of the sugar rings as the methacrylamido groups linked with Cb. The resulting steric hindrance around the methacrylamido reactive sites greatly slowed propagation leading to very slow homopolymerization of TMS-MAG (see Supporting Information for details). Synthesis of Poly(ethylene-alt-propylene)−Poly(DMA-grad-MAG) (PG) Diblock Terpolymers. The glucose-functionalized PG diblock terpolymers were synthesized using a combination of anionic and reversible addition− fragmentation chain transfer (RAFT) polymerizations, as shown in Scheme 2. A hydroxyl-terminated PEP23 was made by anionically polymerizing isoprene in cyclohexane at 40 °C, functionalizing with ethylene oxide at the ω-terminus, followed by hydrogenation using Pt/SiO2 as the heterogeneous catalyst. The Mn (1H NMR, end-group analysis) was 3.2 kg mol−1, and D̵ (SEC) was 1.05 in chloroform relative to PS standards. The polyisoprene precursor contained 91% 4,1 additions of isoprene, and full saturation (>99%, NMR) was achieved in the hydrogenation step. The Tg of PEP-OH was −65 °C, as C
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Table 1. Molecular Characteristics of the Amphiphilic Diblock Copolymers and Terpolymers samplea
NEPb
Namide or NEOb
MAG (mol %)c
PEP (wt %)d
Mn (kg/mol)e
D̵ f
PG(3-26-0.39) PG(3-24-0.21) PG(3-24-0.16) PG(3-19-0.08) PA(3-21) PO(3-25)h
45 45 45 45 45 45
165 181 192 176 208 565
39 21 16 7.8 0 0
11 12 12 14 13 11
29 27 27 23 24 28
1.31 g 1.29 1.18 1.15 1.02
a PG: PEP−poly(DMA-grad-MAG); PA: PEP−PDMA; and PO: PEP−PEO. The enclosed first two values in PG and those in PA and PO are the number-average molecular weights in kg mol−1 of the component blocks, and the third values in PG samples are the mole fraction of MAG repeating units in the hydrophilic block, as determined by 1H NMR spectroscopy. bDegree of polymerization. cMole fraction of MAG in the hydrophilic block. d Weight fraction of the hydrophobic PEP block. eTotal number-average molecular weight. fAll samples were measured on a SEC using CHCl3 as the eluent at 35 °C and relative to polystyrene standards, except PO(3-25), which was measured on a SEC using THF/N,N,N′,N′tetramethylethylenediamine as the eluent at 25 °C and equipped with a light scattering detector. The values of the PG diblock terpolymers before hydrolysis are reported, which are likely to be overestimates of the true values (see Figure S6 for details). gSEC analysis of this sample was not performed prior to deprotection. Another sample synthesized under similar conditions gave a D̵ = 1.31 (see Table S2). hData reproduced from ref 27.
°C, followed by removal of the trithiocarbonate group as above. PO(3-25)27 was prepared by the anionic polymerization of ethylene oxide in THF at 40 °C. The molecular characteristics of the diblock polymers are summarized in Table 1. Reactivity Ratios of DMA and TMS-MAG in FreeRadical Polymerizations. We explored the copolymerization of DMA (as monomer 1) and TMS-MAG (2, as monomer 2) in free-radical polymerizations to determine the reactivity ratios at 70 °C, as shown in Scheme S2. The solvent was a 1:1 (v/v) mixture of toluene and 1,4-dioxanethe same as that in the RAFT synthesis of the PG diblock terpolymers. We carried out 13 experimental runs with the monomer fractions of DMA in the feed ( f1) ranging from 0.10 to 0.90 and kept the conversion of each monomer less than 15.0%. We determined f1 and the conversion of two monomers by 1H NMR spectroscopy and calculated the mole fractions of DMA in the copolymer (F1) using the f1 and conversion data (Table S3). For run 1 (f1 = 0.905, F1 = 0.939), we also isolated the copolymer product and analyzed its composition by 1H NMR spectroscopy and obtained consistent results (F1 = 0.946). We fit the composition data using the nonlinear method,30 following F1 = (r1 f12 + f1 f 2)/(r1 f12 + 2f1 f 2 + r2 f 22), and determined r1 = 1.86 ± 0.07, r2 = 0.16 ± 0.01, as shown in Figure 1.31 r2 is much smaller than r1 (r2/r1 < 0.1), which is
consistent with the slow homopolymerization of TMS-MAG (page 1, Supporting Information). Methacrylamides typically exhibit higher reactivity than acrylamides, and we ascribe the difference to be the excessive steric hindrance from TMSprotected glucosamine in TMS-MAG (α anomer as well as large TMS groups). Based on these results, the selfpropagations of the terminal TMS-MAG radials are ca. 6 times slower than their cross-propagations toward DMA, which have much less steric hindrance than TMS-MAG monomers. On the other hand, propagating chains with terminal DMA radicals have only ca. 2-fold self-propagation preference. With r1r2 = 0.30 (
