Article Cite This: Macromolecules 2019, 52, 5231−5244
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Rodlike Block Copolymer Micelles of Controlled Length in Water Designed for Biomedical Applications Qing Yu,† Megan G. Roberts,† Samuel Pearce,‡ Alex M. Oliver,‡ Hang Zhou,† Christine Allen,∥ Ian Manners,‡,§ and Mitchell A. Winnik*,†,¶
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†
Department of Chemistry and ¶Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada ‡ School of Chemistry, University of Bristol, Bristol BS8 1TS, U.K. § Department of Chemistry, University of Victoria, Victoria, British Columbia V8W 3V6, Canada ∥ Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College Street, Toronto, Ontario M5S 3M2, Canada S Supporting Information *
ABSTRACT: There is a broad interest in elongated colloids as drug delivery vehicles, and current research aims to address how their length and aspect ratio affect interactions with cells. Block copolymer (BCP) micelles offer the opportunity to vary micelle length while maintaining cross-sectional width with corona chains that maintain a common surface chemistry across these structures. However, most elongated BCP micelles used in cell studies are characterized by a very broad length distribution. Here, we describe the synthesis and self-assembly properties of a diblock copolymer with a polyferrocenylsilane core-forming block and a corona block consisting of a statistical polymer of (aminopropyl)methacrylamide and oligo(ethylene glycol methacrylate) (M = 500) (PFS27-bPAPMA3-stat-OEGMA48). Self-assembly in water gave a mixture of structures including rodlike micelles. In alcohols, different types of structures were obtained depending on the alcohol employed (butanol, 2-propanol, ethanol, and methanol). In ethanol, the polymer formed long micelles of uniform width by crystallization-driven self-assembly. Following sonication, a series of rodlike micelles with different lengths (80 to 2000 nm) and narrow length distributions (Lw/Ln < 1.10) were generated by seeded growth. These micelles could be transferred to aqueous media and maintained colloidally stable in PBS (phosphatebuffered saline) buffer for more than three months. In these micelles, the POEGMA brush provides a “stealth” coating to minimize the interaction with proteins and cells, and the APMA groups provide functionality for attachment of drugs or metal chelators for potential therapeutic applications. Studies in two human breast cancer cell lines (MDA-MB-231 and MDA-MB436) show no signs of toxicity for micelle concentrations up to 0.1 mg·mL−1. We also show that metal chelators can be covalently attached to the amino groups in the corona and labeled with heavy metals, opening the door to future experiments with radionuclides.
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gold nanorods (NRs)12 and CdSe NRs,13 and more flexible structures such as wormlike linear aggregates of iron oxide nanoparticles,14 bottlebrush polymers,15−17 and various cylindrical block copolymer (BCP) micelles. Others have looked at thicker structures such as stretched polystyrene particles,18 silica nanorods and polymer capsules templated by these nanorods,19 and hydrogel and solid nanoparticles fabricated by PRINT technology.20−23 One of the advantages of amphiphilic elongated BCP micelles is that the surface chemistry is determined by the water-soluble polymer block that forms the micelle corona. For elongated micelles, the corona forms a dense brush covalently grafted to the core-forming polymer and can provide functionality for attaching targeting agents. As in the many
INTRODUCTION Interest in elongated nanoparticles for drug delivery dates back to the early 2000s. Early experiments examining biodistribution and cell uptake of carbon nanotubes1,2 were followed by an important series of papers by Discher’s group on long (5 to 20 μm), flexible cylindrical block copolymer (BCP) micelles with a low glass-transition temperature (Tg) core-forming polymer (polyethylethylene or polycaprolactone) and poly(ethylene glycol) as the corona block.3−5 Each micelle sample was characterized by a very broad length distribution.5,6 To emphasize their flexibility and draw analogy to filamentous viruses, the authors referred to these structures as filomicelles. Over the ensuing years, there has been a broad recognition that size, shape, and surface chemistry are key factors that can affect cell uptake, tumor penetration, and blood circulation time, although the optimum combination of these criteria are yet to be established.7−11 Among the elongated objects examined for their biological interactions are thin rigid structures such as © 2019 American Chemical Society
Received: May 8, 2019 Revised: June 18, 2019 Published: July 8, 2019 5231
DOI: 10.1021/acs.macromol.9b00959 Macromolecules 2019, 52, 5231−5244
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nm and 450 ± 210 nm) in which the low Tg of the PBA generates a liquid-like micelle core.30 In this way, they could examine how the stiffness of the micelle affected the interaction with cell lines and multicellular tumor spheroids of cells that overexpress the GLUT5 transporter. These experiments address another issue in the design of drug delivery vehicles, namely, how the stiffness or elasticity of a nanoparticle drug carrier affects its uptake by cells and its penetration into tumors.30 In this discussion, “stiffness” refers to the compression modulus of the nanoparticle as determined by AFM rather than the persistence length of an elongated micelle.31 In the examples reported by Stenzel’s group, the micelles with the glassy PMMA core were characterized by a higher modulus (∼2500 MPa) than the micelles with the low Tg PBA core that collapsed on the substrate in the dry state (∼350 MPa). The stiffer micelles showed higher uptake by two different cell lines. In the Discher group’s studies, the filomicelles were sufficiently long that their length and shape could be imaged by video microscopy. Through analysis of these images, they evaluated the persistence length (lp) of these structures and obtained lp ≈ 500 nm for the micelles with a core diameter of 11 nm and lp ≈ 5 μm for the micelles with a core diameter of 29 nm.5 In spite of the liquid-like low Tg core, these micelles were characterized by large lp values, a feature that can be attributed to both core thickness and repulsive interactions of the corona chains. Corona repulsion is also the origin of the conformational and shape-persistent properties of bottlebrush polymers.15 In this paper, we describe the design, preparation, and characterization of a family of rodlike BCP micelles of controlled length and narrow length distribution for eventual use as nanoparticle delivery agents. In this example, we employ a diblock copolymer with polyferrocenyldimethylsilane (PFS) as the crystalline-core-forming polymer. The corona block is a statistical copolymer of oligo(ethylene glycol methacrylate) and (aminopropyl)methacrylamide (N3-PAPMA3-stat-POEGMA48), where the POEGMA confers colloidal stability and is intended to provide stealth properties for future in vivo experiments.32,33 The amine groups of the APMA monomers can serve as sites for drug attachment or, for the application envisioned here, as sites of attachment of metal chelators for radiometals for radioimmunotherapy applications.34 Self-assembly of this BCP was examined in several different alcohol solvents. Uniform rodlike micelles could be prepared in ethanol. A series of rodlike micelles with different lengths (80, 200, 500, 1000, and 2000 nm) and narrow length distributions were prepared via seeded growth. These micelles were easily transferred from ethanol into water. They were colloidally stable in phosphate-buffered saline (PBS) buffer and remained stable for more than several months. The micelle dimensions were characterized by multiangle light scattering and TEM. As a proof-of-concept experiment, we attached DTPA metal chelators to the amine groups of the preformed micelles and used Tb3+ ions to demonstrate their ability to bind heavy metals.
examples of spherical micelles as drug delivery vehicles, the amorphous polymer that makes up the micelle core can serve as a host to dissolve or otherwise carry drug molecules in an attempt to deliver them to their intended target.7 Cylindrical micelles with a core consisting of an amorphous polymer tend to be uniform in their core cross-sectional width but rather polydisperse in length. For example, Wooley’s group prepared short cylindrical micelles (length L = 180 ± 120 nm; core width W = 20 ± 2 nm) with a polystyrene core and a poly(acrylic acid) corona24 (PAA96-b-PS48, the subscripts refer to the mean degrees of polymerization) and longer and thicker micelles (L = 900 ± 180 nm; W = 30 ± 2 nm) prepared from a PAA94-b-PMA103-b-PS28 triblock terpolymer (PMA = poly(methyl acrylate)).24,25 The PAA core of both types of micelles were then cross-linked by reacting with a diamine followed by introducing a targeting agent. Elongated micelles can also be prepared by polymerizationinduced self-assembly (PISA). In this technique, one prepares a macromonomer from the corona-forming block and then uses it to initiate polymerization of a second monomer that will form the micelle core.26,27 Microphase separation takes place during the reaction, leading to rodlike structures once the insoluble block reaches a critical length. Kaga et al.8 used PISA to prepare nanoparticles from a family of poly(glycidyl methacrylate)-b-poly(oligo(ethylene glycol) methyl ether methacrylate)-b-polystyrene (PGMA9-b-POEGMA26-b-PSn, n = 135, 173, 280, and 352) triblock terpolymers. The polymers with PS135 and PS173 formed spherical micelles, whereas those with PS280 and PS352 formed elongated micelles. The authors used the epoxy groups of the PGMA block to introduce a radiolabel for biodistribution studies and then converted the remaining epoxy groups to vicinal diols. The PS280 micelles had contour lengths of 350−500 nm (W = 38 ± 4 nm). They had a kinked curvilinear shape, perhaps reflecting the rigidity of the glassy PS core. The authors refer to these shorter structures as “rods”. The PS352 micelles had contour lengths of 1−2 μm (W = 45 ± 4 nm). Due to the long entangled networks they observed for the longer micelles, they refer to them as “worms”. In a subsequent paper, micelles generated by PISA were used by Gooding et al. to examine shape effects on subcellular localization in cell uptake experiments.28 Stenzel’s group reported a series of elongated block copolymers with a poly(methyl methacrylate) core and a fructose-based methacrylate corona.29,30 In one interesting example, the group used nanoprecipitation at various stirring rates (100, 500, and 1000 rpm) to produce elongated micelles of P(1-O-MAFru)31-b-PMMA166 in which the mean micelle length decreased with increasing stirring rates.29 Very long micelles (∼2 μm) were obtained at the slowest stirring rate (100 rpm), and micelles with mean lengths of 800 and 400 nm were obtained at intermediate and higher stirring rates (500 and 1000 rpm). They examined the interaction of these micelles with cell lines that overexpress GLUT5 transporters, which interact with fructose. These micelles were found to have a broad contour length distribution. In transmission electron microscopy (TEM) images, the micelles have a curved contour and are accompanied by a small fraction of other shapes, particularly spheres and toroids. The PMMA core is presumably in its glassy state at 25 °C and, as in the case of PScore micelles, these micelles are likely to be locally rigid. In a later paper, they compared these micelles (here L = 640 ± 240 nm and 1420 ± 570 nm) with those prepared from P(1O-MAFru)31-b-PBA158 (BA = butyl acrylate) (L = 310 ± 120
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EXPERIMENTAL SECTION
Instrumentation. Nuclear magnetic resonance (NMR) spectroscopy experiments were performed on an Agilent DD2 600 spectrometer using a 10 s delay time and 45° pulse angle at room temperature. Gel permeation chromatography (GPC) measurements were conducted on two different instruments. PFS homopolymers were 5232
DOI: 10.1021/acs.macromol.9b00959 Macromolecules 2019, 52, 5231−5244
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Macromolecules analyzed using a Viscotek GPC MAX liquid chromatograph equipped with a TDA302 triple detector array and a Viscotek 2501 UV detector (set at 450 nm to detect PFS). The column temperature was kept at 36 °C. The GPC was calibrated using polystyrene standards, and THF flowing at a rate of 1 mL/min was used as the eluent. To analyze PFS-b-PAPMA-stat-POEGMA and N3-PAPMA-stat-POEGMA polymers, a salt THF solution, containing 0.25 g/L tetra-n-butylammonium bromide (TBAB) at a flow rate of 0.6 mL/min, was used as the eluent. The salt THF GPC measurements were performed using a Waters 515 HPLC equipped with a Viscotek VE 3580 RI detector and a 2500 UV/Vis detector. It was calibrated against poly(methyl methacrylate) standards. Matrix-assisted laser desorption ionization time-of-light (MALDITOF) experiments were carried out using an Applied Biosystems 4700 Proteomics analyzer operating under the reflector mode. trans-2[3-(4-tert-Butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) dissolved in THF (20 mg/mL) was used as the matrix. The PFS homopolymer in THF (10 mg/mL) was mixed with the matrix at a ratio of 10:1 (v/v). A trace amount (5 μm), uniform rodlike micelles of uniform width (12 ± 2 nm). Crystalline BCP micelles prepared by rapid cooling of hot BCP solutions may sometimes exhibit a complicated micelle morphology.50 This may explain in part the complex morphologies formed in n-BuOH and 2-PrOH. However, other factors are more likely to play an important role here. The various micelle morphologies observed above can be attributed in part to the different solubilities of PFS in these selective solvents. Good solvents for PFS retard nucleation51 and also slow down the micelle growth rate.52 Poor solvents for the corona chains are predicted to reduce the growth rate because the smaller dimensions of this block screens access of the incoming BCP to the growing end of the micelle.53 The solubility parameters (δ) of the polymers and solvents are collected in Table 3. Methanol (δ = 29.6 MPa1/2) is the poorest solvent for both PFS (δ = 18.7 MPa1/2)54 and POEGMA (δ = 19.3 MPa1/2),55 and this may reduce the
Table 3. Solubility Parameters of Polymers and Solvents solubility parameters (MPa1/2) polymers or solvents
δDa
δPa
δH a
δa
PFS POEGMA MeOH EtOH 2-PrOH BuOH H2O
15.4 15.2 15.8 15.8 16.0 15.6
7.6 12.3 8.8 6.1 5.7 16.0
8.7 22.3 19.4 16.4 15.8 42.3
18.7b 19.3 29.6 26.6 23.6 23.2 47.8
Hansen solubility parameters:59 δD, dispersion parameter; δP, dipolar parameter; δH, hydrogen bonding parameter; δ, total solubility parameter. bHildebrand solubility parameter.54 a
propensity to form rodlike micelles. Lenticular micelles have previously been reported for other PFS BCPs including PFS-bP2VP56−58 [P2VP = poly(2-vinylpyridine)] and PFS-b-PP57 (PP = poly[bis(trifluoroethoxy)phosphazene]). In those examples, the authors explained that the lenticular micelles exhibit polycrystalline cores, and the shape was a result of crystallization in both the lateral and longitudinal directions. Moreover, the irregular crystal growing fronts were explained as the consequence of defects caused by interfering corona blocks. These lenticular shapes are often seen in PFS BCPs that have relatively rigid corona blocks such as P2VP and PP and rarely observed in PFS BCPs having flexible coronaforming blocks such as PI and PDMS. Based on these observations, we believe that the formation of lenticular structures in methanol can be attributed to the poor solvent quality of methanol for PFS, and the limited flexibility of the POEGMA brush polymer in the PFS27-b-PAPMA3-stat5238
DOI: 10.1021/acs.macromol.9b00959 Macromolecules 2019, 52, 5231−5244
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Figure 5. (a, b) TEM images and length distribution histograms of short micelle fragments obtained by sonication: Ln = 81 nm, Lw = 91 nm, Lw/Ln = 1.12, and σ/Ln = 0.30. Scale bar is 200 nm. (c−j) TEM images and length distribution histograms of PFS27-b-PAPMA3-stat-POEGMA48 micelles obtained from seeded growth experiments in ethanol: (c, g) Ln = 197 nm, Lw = 203 nm, Lw/Ln = 1.03, and σ/Ln = 0.18; (d, h) Ln = 497 nm, Lw = 510 nm, Lw/Ln = 1.03, and σ/Ln = 0.15; (e, i) Ln = 978 nm, Lw = 1002 nm, Lw/Ln = 1.02, and σ/Ln = 0.15; (f, j) Ln = 1835 nm, Lw = 1869 nm, Lw/ Ln = 1.02, and σ/Ln = 0.13. Micelle solutions were diluted to 0.1 mg/mL for TEM measurements. All scale bars are 500 nm.
Preparation of Rodlike Micelles of Controlled Lengths. Starting with rodlike micelle samples prepared in ethanol, we employed the seeded growth method to prepare micelles of controlled length and narrow length distribution. Long micelles obtained from PFS27-b-PAPMA3-stat-POEGMA48 in ethanol were sonicated at room temperature for 30 min using a 70 W ultrasonic cleaning bath. The resulting short micelle fragments had Ln = 81 nm, Lw = 91 nm, Lw/Ln = 1.12, and σ/Ln = 0.30 (Figure 5a,b). To prepare rodlike micelles with target lengths of 200, 500, 1000, and 2000 nm, different amounts of unimer solution (10 mg/mL PFS27-b-PAPMA3stat-POEGMA48 in THF) were added to the short micelle fragments solution in EtOH. The mixtures were aged at room temperature for 1 week. TEM images of the micelles obtained in this way are presented in Figure 5c−f. Length distribution histograms were constructed by measuring 200 micelles for each sample (Figure 5g−j). The Ln values obtained are in good agreement with the targeted lengths, and all the rodlike micelles from seeded growth exhibit narrow length distributions, with the widest distribution associated with the seed fragments generated by sonication. Light Scattering Studies. Rodlike PFS27-b-PAPMA3-statPOEGMA48 micelles in ethanol were examined by multiangle
POEGMA48 micelles as well as the poor solvent quality of methanol for the corona chains. Ethanol, 2-propanol, and n-butanol are increasingly better solvents for both PFS and POEGMA. In these solvents, rodlike micelles are prominent, although in 2-propanol and n-butanol, they are accompanied by thin planar platelet-like structures. At this time, we have no unique explanation for these platelets. Because the corona chains in our sample have a somewhat broad dispersity (Đ = 1.3), it is possible that the platelets are formed selectively and more rapidly by the polymer fraction with the shortest corona block, but we have no evidence for this kind of sample fractionation. We do note that BCP molecules with longer corona chains tend to add more slowly to the growing micelle than those with shorter corona chains.60 As a test of the idea that dispersity can play a role in the selfassembly, we examined the self-assembly of two similar polymers prepared in the absence of APMA (see Figures S8−S10, Supporting Information for details). Both PFS20-bPOEGMA90 (Đ = 1.09) and PFS30-b-POEGMA90 (Đ = 1.11) form long cylindrical micelles of uniform width in 2-PrOH. Nevertheless, the key point is that none of these complications are seen for the self-assembly of PFS27-b-PAPMA3-statPOEGMA48 in ethanol. 5239
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Figure 6. (a) SLS data of PFS27-b-PAPMA3-stat-POEGMA48 micelle fragments in ethanol, represented as Holtzer−Casassa plots of qRθ/πM0Kc as a function of q and (b) DLS data (plots of Γ1/q2 as a function of qL, where L corresponds to SLS Lw values) for the same micelle fragments. The red data points and lines refer to short micelle fragments of Ln = 242 nm, and the blue ones refer to long micelle fragments of Ln = 605 nm. These are the same samples shown in the TEM images in Figure S11.
RDLS = 15 nm, and the longer micelles had RDLS = 14 nm. These values are larger than RSLS, as expected. The value of RSLS is dominated by the dense core of the micelles, whereas RDLS is strongly influenced by the corona chains that extend from the core into the solution. The difference between them is small because of the short length of the corona chains (DP ≈ 50). In ref 45, we compared the values of RDLS for PFS26-bPOEGMA163 in methanol, methanol/ethanol (1:4, v/v), and water. While the values were similar in the two alcoholic media (22 nm), we found a small increase in water (28 nm). Thus, we expect a small increase in RDLS for PFS27-b-PAPMA3-statPOEGMA48 after transfer to water. Transfer of Rodlike Micelles to Water. The rodlike micelles prepared in ethanol were transferred into water as described in the Experimental Section. Compared with the commonly used dialysis method, which takes a few days, our solvent exchange method using centrifugal filters can shorten this process to less than an hour. As shown by TEM images in Figure 7a,b, no significant change in the morphology of the micelles (e.g., fragmentation)63 was observed after solvent exchange. An example is shown in Figure 7a,b for a sample with Ln = 978 nm before transfer and Ln = 960 nm after transfer to water. More importantly, the length distribution histograms (Figure 7c−f) showed that the micelles maintained their narrow length distribution in water. In Table S2, we collect values of Ln, Lw, and σ/Ln for all of the micelle samples prior to and after transfer to water. Since these rodlike micelles were synthesized for biomedical applications, their colloidal stability in biological environments is a critical factor and needs to be estimated prior to cell studies. To do so, we transferred the rodlike micelles to PBS buffer (pH 7.4) using the spin filtration method and stored them at 4 °C in the dark for 3 months. The micelle solutions were analyzed by DLS and TEM. DLS CONTIN plots show symmetric and narrow distribution for all five samples (Figure S13, Supporting Information), and no significant change in the morphology of the micelles was detected by TEM (Figure S14, Supporting Information). No aggregation or degradation were detected in either measurement, indicating that these rodlike micelles exhibited good stability in PBS buffer for at least 3 months. Cell Viability Tests. Since our intent is to use these rodlike micelles in cell uptake and multicellular tumor spheroid penetration studies, it is essential that we examine the toxicity of the micelles to cells in cell culture. Here, we use the MTS cell viability assay64 to examine the toxicity of the micelle
static and dynamic light scattering (SLS and DLS) to characterize their shape in solution.45,61 Two micelle samples of different lengths were prepared via seeded growth in ethanol (Figure S11) and diluted to c = 0.02 mg/mL to ensure that the structure factor would be negligible. The data were fitted to a model of rigid rods of finite thickness. The SLS results are represented by Holtzer−Casassa (HC) plots (Figure 6a) showing qRθ/πMoKc as a function of q, where q is the scattering vector, Rθ is the Rayleigh ratio, Mo is the polymer molecular weight, c is the concentration, and K is an optical constant. The excellent agreement seen in Figure 6a between the data and the fitted curves emphasize that these micelles are rigid rods in shape. We obtained three important parameters from the HC plots: (i) the weight-average length (Lw) of the micelle, (ii) the radius of the cylinder’s cross section, RSLS, and (iii) the linear aggregation number Nagg/L, which is the number of BCPs per unit length of the rigid rodlike micelles. As shown in Table 4, Table 4. Structural Parameters Characterized by TEM and Light Scattering of Micelles in EtOH type of micelles short micelles long micelles
LwTEM (nm)
LwSLS (nm)
Nagg/L (chains/nm)
RSLS (nm)
RDLS (nm)
249
270
4.7
10
15
644
670
5.1
10
14
the HC plots yielded Lw values in good agreement with those obtained from TEM, suggesting that our data analysis is valid. The cylinder’s cross section of both short and long micelle fragments had a radius RSLS of 10 nm. This value is a measure of the mass distribution of both the core polymer and the corona chains perpendicular to the long axis of the micelles. Most importantly, the HC plots of rodlike PFS27 -bPOEGMA48 micelles in ethanol have Nagg/L = 4.7−5.1 chains/nm. These dimensions are consistent with results that we reported previously with a related BCP, PFS26-bPOEGMA163, where the POEGMA composition was designed to exhibit an LCST in water at about 40 °C.45 For that polymer in methanol/ethanol = 1:4 (v/v), we found Nagg/L = 5.4 chains/nm and RSLS = 10 nm. The hydrodynamic radius cross section, R DLS , was determined from the DLS data (Figure 6b) using a model derived by Wilcoxon and Schurr.62 The shorter micelles had 5240
DOI: 10.1021/acs.macromol.9b00959 Macromolecules 2019, 52, 5231−5244
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Figure 7. TEM images of micelles (Ln = 978 nm) in (a) ethanol at 0.05 mg/mL and (b) after transfer to water. (c−f) Length distribution histograms of the micelles in water. (c) Ln = 188 nm, Lw = 202 nm, Lw/Ln = 1.07, and σ/Ln = 0.27. (d) Ln = 475 nm, Lw = 490 nm, Lw/Ln = 1.03, and σ/Ln = 0.17. (e) Ln = 960 nm, Lw = 989 nm, Lw/Ln = 1.03, and σ/Ln = 0.17. (f) Ln = 1823 nm, Lw = 1896 nm, Lw/Ln = 1.04, and σ/Ln = 0.20. Scale bars: 1000 nm.
sample with Ln = 978 nm in two different human breast cancer cell lines (MDA-MB-231 and MDA-MB-436). The cells were incubated with a series of micelle concentrations for 24 h at 37 °C. The results in Figure 8 showed that micelles are nontoxic to both cell lines up to a concentration of 0.1 mg/mL. Covalent Modification of the Micelle Corona with DTPA. One of the possible applications of the rodlike micelles prepared in this study is as a carrier of radionuclides for radioimmunotherapy applications. As illustrated in Scheme 3,
preparation of the metal-chelating micelles took place in two steps. In the first step, rodlike micelles of Ln = 81 nm were reacted with DTPA in water at pH 8.5 in the presence of the peptide-coupling agent DMTMM. We used a large excess of DTPA pretreated with DMTMM (80 equiv based on polymer amine groups). These reaction conditions are very effective at adding DTPA to pendant amino groups of free polymers in aqueous solutions,34,65 but this is our first attempt to use this reaction on functional groups buried in the dense brush corona of a micelle sample. In the second step, the DTPA groups were labeled with Tb3+ in citrate buffer (pH 6) for 1 h at room temperature. The resulting Tb3+-PFS27-b-PAPMA3-stat-POEGMA48 micelles were washed extensively with water using a centrifugal spin filter to ensure the removal of free chelators and ions. To quantify the amount of Tb3+ ions on the micelles, Tb3+PFS27-b-PAPMA3-stat-POEGMA48 micelles were dissolved and diluted in 2% HNO3 to different concentrations for ICP-MS analysis. The results are summarized in Table S3 and show that there is an average of 0.25 ± 0.02 Tb3+ ions per polymer chain. This result, which is somewhat surprising, indicates that the DTPA coupling/Tb3+ binding step occurs with low efficiency (ca. 10%), whereas with the free polymer in solution (with multiple pendant −NH2 groups per polymer), the reaction is essentially quantitative. This is an aspect of this reaction that deserves further investigation. This observed result here, combined with the micelle’s linear aggregation number (Nagg/L = 5 chains/nm), indicates that, on average, each rodlike micelle (Ln = 81 nm) carried 101 ± 9 Tb3+ ions.
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Figure 8. Cell viability of MDA-MB-231(blue) and MDA-MB-436 (red) cells after exposure to PFS27-b-PAPMA3-stat-POEGMA48 rodlike micelles of Ln = 978 nm for 24 h up to a micelle concentration of 0.1 mg/mL. The data represented as mean ± standard deviation, n = 3.
CONCLUSIONS In summary, we report the synthesis of a polyferrocenylsilanepoly[oligo(ethylene glycol methacrylate)] block copolymer 5241
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Macromolecules Scheme 3. Conjugation of PFS27-b-PAPMA3-stat-POEGMA48 Micelles with DTPA and Labeling with Tb3+ ions
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PFS27-b-PAPMA3-stat-POEGMA48 in which the APMA units contribute ca. three −NH2 groups per polymer. The polymer was synthesized by a combination of anionic polymerization, ATRP, and a CuAAC “click” coupling reaction. Self-assembly experiments showed that the solvent has a major influence on the structures formed. In water, a mixture of rodlike micelles and other ill-formed objects were seen in TEM images, whereas in methanol, lenticular micelles were formed. In butanol and 2-propanol, we observed “scarf-like” micelles with a platelet-like central region with cylindrical micelles protruding from the two ends. In ethanol, the self-assembly was well behaved, yielding long (>5 μm) fiber-like micelles of uniform width. Following sonication to obtain micelle fragments as seed micelles, we prepared a family of rodlike core-crystalline BCP micelles of uniform width and narrow length distribution, with number-average lengths ranging from 80 to 2000 nm. The micelles could be transferred to aqueous solutions and are colloidally stable over long periods of time (months) in water and PBS buffer. The intended application for these micelles is as potential drug delivery vehicles or carriers of radionuclides for radioimmunotherapy. As steps in this direction, we used the MTS cell viability assay to show that the micelles are nontoxic to two breast cancer cell lines (MDA-MB-231 and MDA-MB436) at concentrations up to 0.1 mg/mL. In addition, we showed that DTPA as a metal chelator could be linked to the −NH2 groups in the corona brush of the micelle and labeled with Tb3+ as a representative heavy metal ion. While these micelles do not carry functionality for attachment of antibodies or other bioaffinity agents, this functionality can, in principle, be incorporated by coassembly of PFS27-b-PAPMA3-statPOEGMA48 with a second PFS block copolymer (for example, with a heterobifunctional PEG) containing a reactive group at the distal end of the corona chain. Experiments examining cell uptake and penetration into model tumor spheroids with these micelle samples are in progress and will be reported in the near future. In this way, we hope to develop a deeper understanding of how the length of a rigid rod nanocarrier with common surface chemistry affects these two important properties of nanomedicine.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.macromol.9b00959.
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Additional experimental details, characterization data, analysis of light scattering data, and supporting tables and figures (PDF)
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Megan G. Roberts: 0000-0003-1819-7502 Samuel Pearce: 0000-0003-2661-2702 Hang Zhou: 0000-0003-4284-9654 Christine Allen: 0000-0002-4916-3965 Ian Manners: 0000-0002-3794-967X Mitchell A. Winnik: 0000-0002-2673-2141 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The Toronto authors thank NSERC Canada for their support of this research. Q.Y. and M.R. thank Ms. Loujin Houdaihed for the help with cell viability experiments. The authors also thank the CSB and the CPO at the University of Toronto for the use of their imaging facilities.
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