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Article Cite This: J. Am. Chem. Soc. 2017, 139, 14684-14692
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“Sweet” Architecture-Dependent Uptake of Glycocalyx-Mimicking Nanoparticles Based on Biodegradable Aliphatic Polyesters by Macrophages Libin Wu,† Yufei Zhang,† Zhen Li,† Guang Yang,† Zdravko Kochovski,‡ Guosong Chen,*,† and Ming Jiang† †
The State Key Laboratory of Molecular Engineering of Polymers and Department of Macromolecular Science, Fudan University, Shanghai 200433, China ‡ Institute of Physics, Humboldt University of Berlin, Newton Strasse 15, 12489 Berlin, Germany S Supporting Information *
ABSTRACT: Glyconanoparticles made by self-assembled glycopolymers currently are practical and efficient mimics of the glycocalyx on cell surfaces. Considering the complexity of the glycocalyx, glyconanoparticles with different sugars on their coronas, i.e., mixed-shell glycomicelles, could be more valuable compared to homoshell micelles. In this paper, we explore the architectural effect of the glyconanoparticle corona on glyconanoparticle macrophage endocytosis and lectinbinding ability. A series of glyconanoparticles composed of a biodegradable polyester backbone functionalized with galactoside or mannoside pendants were designed and prepared. The different architectures explored were single-component (galactoside or mannoside) coronas, homogeneously mixed coronas (MG) made by galactoside−mannoside copolymer chains, and blend-mixed coronas (M/G) constructed from two homoglycopolymers. Nanoparticles with a mixed shell showed a higher efficiency in cellular uptake and lectin-binding than those with a single sugar component. Meanwhile, unexpectedly, MG presented a significantly higher efficiency than M/G, although they had the same particle size and ratio of mannoside to galactoside. We attributed this apparent architectural effect to the difference in the phase behavior between MG and M/G; i.e., the former having a homogeneous corona allowed more sugar− receptor interactions in the contact region, while the latter having phase separation limited the simultaneous interaction of the two kinds of sugar units with the cell receptors.
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INTRODUCTION The tremendous heterogeneity of glycans is one of the major obstacles toward understanding the glycocalyx,1 the heavy layer of carbohydrates on cell surfaces that is comprised of glycoproteins, glycolipids, and proteoglycans.2 The glycocalyx plays crucial and complex roles in cell−cell recognition, communication, and intercellular adhesion.3 Step by step, scientists have discovered that these functions are dependent not only on the chemical structure of the glycan chain end sugar units, but also on the physical properties of the polymeric glycan chain, e.g., chain length and rigidity.3b,4 Therefore, we believe self-assembled nanomaterials made by glycopolymers5 are one of the best nano-objects to study the glycocalyx through mimicking the properties of their glycans. More specifically, both the physical properties of the polymer chain and chemical structure of sugars can be tuned. Recently, glyconanoparticles6,7 assembled from glycopolymers have been prepared to understand carbohydrate−protein interactions in solution and on the cell surface. 8 Moreover, different applications of this type of material have been achieved, for © 2017 American Chemical Society
example, as delivery vehicles for drugs, proteins, or oligonucleotides.9 By using glycopolymers, we are able to take the significant advantage of flexibility and convenience in designing different corona architectures for further study on the interactions of these nanomaterials with proteins and cells as a function of the architecture.10 This architecture-dependent study is certainly demanding due to the above-mentioned heterogeneity of glycans. It is known that the native glycan chains are composed of various types of sugars with different compositions; thus, the micelle architecture provides a practical and simplified model system.11 Although attention has been paid to the micelle architecture by polymer scientists12 as an important factor controlling phase separation of micelle coronas and their interaction with polyelectrolytes etc., as far as we know, the same depth of study has not been performed on glycopolymeric micelles so far. Received: August 1, 2017 Published: September 26, 2017 14684
DOI: 10.1021/jacs.7b07768 J. Am. Chem. Soc. 2017, 139, 14684−14692
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Journal of the American Chemical Society
with a multiangle light scattering detector) as 9.1 and 8.5 kDa, respectively (Table 1; Figures S4−S7, Supporting Information;
In this paper, glyconanoparticles with various corona architectures assembled by glycopolymers containing mannoside (Man, M) and/or galactoside (Gal, G) on aliphatic polyesters were prepared as a simplified model of the heterogeneous glycocalyx structure. The biocompatible polyester backbone was prepared through ring-opening polymerization catalyzed by Sn(OTf)2, and then the backbone was saccharide-functionalized through Cu(I)-catalyzed azide−alkyne Huisgen cycloaddition. Both metal catalysts used in preparing our glycopolymers have been used in established biomedical applications; more specifically, Sn(II) is used in the manufacture of the anticancer drug Zoladex from Astra Zeneca, which is a polyester biodegradable nanoparticle that has been in the clinic for over 30 years, and Cu(I)-catalyzed click chemistry is commonly used to prepare biomedically applicable polymers.13 This synthetic strategy allowed for the synthesis of homoglycopolymers and heteroglycopolymers with precisely measured sugar numbers on the same polymeric backbone. Then mixed-shell micelles with Man and Gal on the surface were constructed by either coassembly of their own homoglycopolymers or assembly of the glycopolymers that contained both Man and Gal pendants, while the ratio of the two kinds of sugars on the micelle surface remained identical. We then investigated the interactions of these glyconanoparticles with macrophages14 and related model proteins as a function of the micelle architecture to elucidate the architectural effect of the micelle corona on the function of glyconanoparticles and further understand the nature of glycocalyx. We found that nanoparticles formed by Man- and Gal-containing glycopolymers gave much higher endocytosis efficiency in interacting with macrophages than those formed by blending of two homoglycopolymers. This phenomenon was further analyzed by a combination of different methods that led us to conclude that the architecture of mixed-shell micelles had a significant effect on the function of these micelles.
Table 1. Characterization of Aliphatic Polyesters polyester
Mn (kDa)
Mwa (kDa)
Mw/Mn
no. of AVLsb
PCL PCL-b-PAVL PCL-co-PAVL
5.7 7.4 7.0
6.3 9.1 8.5
1.10 1.23 1.21
9 7
a
The absolute molar mass was measured by GPC-MALS, and the corresponding dn/dc was measured by a refractive index (RI) detector. b The number of AVLs was calculated by 1H NMR.
the dn/dc data are shown in Figure S8, Supporting Information). Then 1H NMR was employed to determine the number of AVLs on each chain for click efficiency calculation. First, the number of CL repeating units of the first block PCL of PCL-b-PAVL was calculated by comparing the integration of the signal at δ 4.14 (−OCH2CH3, from ethanol) with those at δ 2.36 and 4.10 (from CL) to be 23 CL repeating units on the chain (Figure S4). After chain extension of PAVL, comparison of the integration of signals at δ 2.36 and 4.10 (from CL) with that at δ 2.04 (proton of alkyne group on AVL) showed nine AVL units on the second block (Figure S5). Similarly, PCL-coPAVL showed seven AVL units (Figure S6) on the random copolymer. Preparation of Monosaccharide-Modified Aliphatic Polyesters. Monosaccharide-modified polyesters were synthesized through clicking 2′-azidoethyl-O-D-mannopyranoside and/or 2′-azidoethyl-O-D-galacopyranoside onto the alkynecontaining polyester precursors. The block copolymer PCL-bPAVL (BP) was clicked with 2′-azidoethyl-O-D-mannopyranoside or 2′-azidoethyl-O-D-galacopyranoside to obtain block glycopolymers P-BP-Man and P-BP-Gal, respectively. Clicking the azido mannnoside and galactoside simultaneously led to PBP-MG, in which the Man and Gal pendants distribute along the sugar block randomly. Meanwhile random glycopolymer PCL-co-PAVL (CP) was modified via the same method, resulting in P-CP-Man, P-CP-Gal, and P-CP-MG. These reactions were highly efficient as supported by their relative 1 H NMR spectra (Scheme 1; Figures S9−S14, Supporting Information). It is natural to presume the azido-functionalized sugars have the same reactivity, while normally it is hard to differentiate the modifications from different sugars on the same polymer chain. Fortunately, the clear 1H NMR spectra of the current aliphatic polyester provided an opportunity to calculate the modification ratio of Man to Gal on P-BP-MG and P-CP-MG. The magnified peaks of triazole ring protons after click reactions are shown in Figure 1. It is obvious that attaching Man or Gal to the polyester chain led to different chemical shifts of the protons of the triazole ring and makes calculating the mole ratio of Man to Gal in P-BP-MG and PCP-MG possible. When α-mannoside was modified onto the polymer chain, the chemical shift of the corresponding triazole was found at δ 7.78, while those of galactoside were found at δ 7.92 (α-anomer) and 7.89 (β-anomer). The ratio of these two peaks was 7.3:2.7, close to the α/β-anomer ratio of 7:3 of 2′azidoethyl-O-D-galacopyranoside. The molar ratio of Man to Gal in P-BP-MG was calculated as 7:10 and that in P-CP-MG as 2:5. The full 1H NMR spectra of the six copolymers are shown in Figures S8−S13 (Supporting Information). Preparation of Glyconanoparticles. To demonstrate the effect of the glycoshell architecture on glyconanoparticle uptake
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RESULTS AND DISCUSSION Design and Synthesis of Biodegradable Aliphatic Polyesters PCL-b-PAVL and PCL-co-PAVL. In our previous study, block copolymers containing a homoglycopolymer block were employed to construct nanoparticles to mimic glycocalyx, and we found that these nanoparticles could convert immunosuppressive macrophages to immunoresponsive ones, showing their great potential in cancer immunotherapy.11 However, these glyconanoparticles were made of nonbiodegradable polymers. Thus, in this paper, we aim at preparing nanoparticles with heterosugar coronas from biodegradable and biocompatible polymers, which of course benefit their further studies in biomedical applications. Aliphatic polyesters characteristic of biocompatibility and biodegradability have attracted broad attention in biomedicine and tissue engineering.15 Therefore, various strategies have been developed in preparing functionalized aliphatic polyesters.16 However, there are few reports on such polymers with sugar pendants.17 In this work, the block copolymer PCL-b-PAVL was prepared through ring-opening polymerization of ε-caprolactone (ε-CL) and αpropargyl-δ-valerolactone (AVL) sequentially using ethanol as an initiator. Furthermore, a random copolymer, PCL-co-PAVL, of the two monomers was obtained through the simultaneous addition of ε-CL and AVL. Both preparations were performed in THF at 60 °C for 24 h and catalyzed by Sn(OTf)2. The absolute molar masses of PCL-b-PAVL and PCL-co-PAVL were determined by GPC-MALS (gel permeation chromatography 14685
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3500). For the block copolymers, the hydrophilic glycoblock and the hydrophobic polyester backbone self-assembled into micelles with the glycoblock as the shell and the polyester as the core. The single sugar micelles BP-Man and BP-Gal were made from the glycopolyesters P-BP-Man and P-BP-Gal, respectively. The blended glycomicelle (BP-M/G) was made by mixing P-BP-Man and P-BP-Gal together at a ratio of 7:10 (number of Man units vs Gal units) to match the ratio in P-BPMG. Accordingly, mixed-shell micelles BP-MG were made from glycopolymer P-BP-MG. The glyconanoparticles were characterized with dynamic light scattering (DLS) and found to share very similar hydrodynamic diameter distributions, i.e., ⟨Dh⟩. The ⟨Dh⟩ of BP-Man was 16 nm (PDI (polydispersity index) = 0.140), that of BP-Gal was 16 nm (PDI = 0.160), that of BPM/G was 15 nm (PDI = 0.197), and that of BP-MG was 16 nm (PDI = 0.244) (Figure 2A). Similarly, glyconanoparticles formed by random glycopolyesters were prepared via the same strategy. Accordingly, glyconanoparticles CP-Man and CP-Gal were prepared from P-CP-Man and P-CP-Gal, respectively, and the blend of these two copolymers led to glyconanoparticle CP-M/G following the same Man/Gal ratio of CP-MG prepared from glycopolyester P-CP-MG itself. DLS demonstrated that these four glyconanoparticles from random copolymers also share similar ⟨Dh⟩ values, namely, (CP-Man) 38 nm (PDI = 0.243), (CP-Gal) 34 nm (PDI = 0.228), (CPM/G) 36 nm (PDI = 0.180), and (CP-MG) 37 nm (PDI = 0.229) (Figure 2B). Their spherical morphology was confirmed by transmission electron microscopy (TEM) with negative staining by uranyl acetate (Figure S15, Supporting Information). The glyconanoparticles made by random copolymers were further characterized by cryo-TEM (Figure 2C,D). The cryo-TEM results showed that the average diameters of CP-M/ G and CP-MG were 26 and 28 nm. This was caused by the hydrophilic saccharides coated on the surface of glyconanoparticles, which have a low differential contrast. Such discrepancies between the hydrodynamic diameter determined by DLS and the average size determined by cryo-TEM have also been previously reported.18 Then two series of glyconanoparticles were obtained. It is worth mentioning that, in this series of nanoparticles made of random copolymers (CP series), although there are no typical core−shell structures, the sugar units are believed to be enriched on the particle surface due to its hydrophilicity. Our particular interest is on the pair of BP-M/G and BP-MG and the pair of CP-M/G and CP-MG (Scheme 1). Within each pair, the two nanoparticles share the same size and the same ratio of Man vs Gal, and their only difference comes from the architecture of their coronas: in BP-MG and CP-MG, Gal and Man units are uniformly distributed because they are made of macromolecular chains of randomly aligned sugar units, while in BP-M/G and CP-M/G, the Gal and Man units exist in the corresponding domains, as they come from different molecular chains and their phase separation usually is unavoidable. Corona Architectural Effect on Interactions of Glyconanoparticles with Macrophages. Macrophages are one type of immune cells that uptake substances such as cellular debris, microbes, and any other external materials.19 The receptors recognizing sugar structures are widely expressed on the surface of macrophages. The macrophage mannose receptor (MMR; cluster of differentiation 206, CD206) is a C-type lectin primarily presented on the surface of macrophages and immature dendritic cells that recognizes terminal mannose,
Scheme 1. Synthetic Routes of Glycopolyesters P-BPSaccharide and P-CP-Saccharide
Figure 1. 1H NMR spectra of the (A, left) BP and (B, right) CP series of monosaccharide-modified aliphatic polyesters.
by macrophages, eight glyconanoparticles with different corona architectures were obtained through self-assembly of glycopolyesters in solution (Scheme 2). Briefly, water was slowly added to a glycopolymer solution in DMSO (5 mg/mL), and then the solution was dialyzed against water for 2 d (MWCO 14686
DOI: 10.1021/jacs.7b07768 J. Am. Chem. Soc. 2017, 139, 14684−14692
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Journal of the American Chemical Society Scheme 2. Illustration of the Self-Assembly of Glyconanoparticles
with only one type of sugar on their surface were internalized by macrophages via these receptors separately.11 RAW 264.7 macrophages were chosen as the model cell line. First, the cytotoxicity of the glyconanoparticles was evaluated through cell viability assays, which showed no obvious cytotoxicity up to 100 μg/mL glyconanoparticles (Figure S16, Supporting Information). Fluorescently labeled glyconanoparticles were created through encapsulating fluorescein during the self-assembly process and then adjusting the fluorescence of all glyconanoparticle samples to the same level (Figure S17, Supporting Information). Then these fluorescence-labeled glyconanoparticles were incubated with RAW 264.7 macrophages for various time intervals at given concentrations followed by flow cytometric analysis. The endocytosis efficiency of macrophages to these glyconanoparticles is shown in Figure 3. The results showed that the endocytosis amount increased with both increasing time and increasing dose. Unexpectedly, these results also showed a consistent and very interesting phenomenon: The mixed-shell glyconanoparticles BP-MG were internalized by macrophages much more efficiently than not only particles with a single kind of sugar, i.e., BP-Man and BPGal, but also BP-M/G, which contains the same ratio of Man to Gal on the particle surface as BP-MG. Considering the same size of BP-MG and BP-M/G, they are a pair of glyconanoparticles with only a corona architectural difference. However, they differed significantly in their interaction with macrophages. This result was found to be consistent in the random copolymer glyconanoparticle series. Moreover, CP-MG and CP-M/G exhibited a greater discrepancy than BP-MG and BP-M/G. In addition, confocal microscopy was used to investigate the macrophage uptake of various fluorescent glyconanoparticles. After the glyconanoparticles (50 μg/mL) were incubated with macrophages overnight, the internalization of various glyconanoparticles by macrophages was clearly observed (Figure S18, Supporting Information). To understand the endocytosis results better, an endocytosis inhibition experiment was performed to explore the pathway of glyconanoparticles. Extracellular substances can be transported into cells through several pathways, for example, macropinocytosis, clathrin-dependent endocytosis, and caveolindependent endocytosis, which can be selectively inhibited by rottlerin, chlorpromazine, and genistein, respectively. As shown in Figure S19 (Supporting Information), the cellular uptake efficiency of these glyconanoparticles with various shell architectures was mostly affected after pretreatment of chloropromazine. This result indicated that the glyconanoparticles were internalized primarily by clathrin-mediated endocytosis, which is a type of receptor-mediated endocytosis.
Figure 2. ⟨Dh⟩ of (A) BP-based glycomicelles and (B) CP-based glyconanoparticles. Cryo-TEM images of (C) CP-M/G and (D) CPMG (scale bar 200 nm).
N-acetylglucosamine (GlcNAc), and fucose.20 Macrophage galactose-binding lectin (MGL; CD301) is a type II transmembrane glycoprotein that contains a single carbohydrate recognition domain specific for the monosaccharide Gal/ GalNAc.21 Our previous study showed that glyconanoparticles 14687
DOI: 10.1021/jacs.7b07768 J. Am. Chem. Soc. 2017, 139, 14684−14692
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Figure 4. Endocytosis study of M/G and MG glyconanoparticles treated with receptor-blocked macrophages. (A) The CD206 receptor was blocked by anti-CD206, and (B) the CD301 receptor was blocked by anti-CD301. Figure 3. Graphs showing the time dependence (A) and dose dependence (B) of the endocytosis of glyconanoparticles with RAW 264.7 macrophages. Data are shown as the mean ± SEM of three independent experiments. Key: p < 0.05; **, p < 0.01.
results from the macrophage antibody blocking assay were applicable in other types of receptors, we explored the binding of our glyconanoparticles to plant lectins through isothermal titration calorimetry (ITC) and DLS. Concanavalin A (ConA)22 and peanut agglutin (PNA)23 were chosen as model proteins that specifically bind to Man and Gal, respectively. ConA and PNA were mixed together in a 1:1 ratio to mimic the assortment of receptors on a cell’s surface. In ITC, the total heat released when glyconanoparticles bind to proteins was measured in a single-injection experiment where a higher binding heat release corresponds to a higher binding ability. Figure 5A shows the heat release when glyconanoparticles made from the block copolymers (0.3 mmol, calculated by the number of saccharides) were titrated into the solution of ConA
The glyconanoparticles with different kinds of saccharides showed similar inhibition effects when they were treated with chloropromazine. One could find that the particle shell architecture did not show a significant effect on the endocytosis pathway, which is understandable in that all these particles are sharing similar sizes and shapes. The most striking result came from an antibody blocking assay. It is known that receptor CD206 on macrophages has a specific binding ability to Man, while CD301 recognizes Gal. To further compare the interactions of MG and M/G particles with macrophages, blocking antibodies of CD301 and CD206 were chosen to be preincubated with cells before incubation of the nanoparticles. The nanoparticles with different shell architectures, BP-MG and BP-M/G, were supposed to have the same binding ability to CD301 and CD206, because of the same number of Man and Gal units on their surface. The same behavior was also expected for CP-MG and CP-M/G as well. However, as shown in Figure 4, blocking antibodies of CD206 and CD301 inhibited the interaction of BP-MG and CP-MG with macrophages much more significantly than that of BP-M/ G and CP-M/G, respectively. The more efficient inhibition at higher concentrations of antibodies was observed for both CD206 and CD301 receptors. These results indicate that, compared to particles BP-M/G and CP-M/G, BP-MG and CPMG have much higher binding ability to receptors CD206 and CD301 on macrophages, although the numbers of sugars on their surface are compatible, which significantly emphasizes the shell architectural effect discussed in this paper. Corona Architectural Effect on the Interactions of Glyconanoparticles with Plant Lectins. To determine if the
Figure 5. Heat release of glyconanoparticles binding with lectins measured by ITC: (A) BP series, (B) CP series (ConA:PNA = 1:1). 14688
DOI: 10.1021/jacs.7b07768 J. Am. Chem. Soc. 2017, 139, 14684−14692
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Journal of the American Chemical Society and PNA (molar ratio 1:1, 6.25 μmol) in HEPES buffer (10 mM, with 1 mM Ca2+ and 1 mM Mn2+). It was found that BPMG gave a higher heat release than BP-M/G, BP-Man, and BPGal, which is similar to the trend we observed in the endocytosis experiment. This result indicates that BP-MG has a higher lectin binding ability compared to BP-M/G, which confirms the result obtained from antibody blocking assay. Similarly, CP-MG released more heat than CP-M/G when they were bound to ConA and PNA (Figure 5B). ConA and PNA are tetramer proteins with four sugar binding sites on each protein moiety; therefore, the lectin−sugar binding induces aggregation of glyconanoparticles that can be measured by DLS. Addition of the ConA and PNA mixture to the solutions of BP-MG and CP-MG induced a faster increase of scattered light intensity (Is/I0) than that of BP-M/G and CPM/G (Figure S20, Supporting Information) respectively, supporting the ITC results. Summarily, BP-MG and CP-MG bound more lectins like ConA and PNA than BP-M/G and CPM/G, respectively, despite the blend glyconanoparticles having a compatible number and ratio of Man and Gal similar to the macrophage endocytosis results. Corona Architectural Effect on Macrophage Activation by Glyconanoparticles. Macrophages can be activated after uptake of external materials, followed by cytokine release in a tightly orchestrated manner, resulting in an inflammatory response.24 As mentioned above, BP-MG and CP-MG have a higher endocytosis efficiency compared with their corresponding M/G glyconanoparticles. Here macrophage activation caused by M/G and MG glyconanoparticles was evaluated. RAW 264.7 macrophages were incubated with glyconanoparticles for 24 h, and the gene expression of cytokines was analyzed using qPCR (real-time quantitative polymerase chain reaction detecting system). The gene expression of two characteristic cytokines, arginase 1 and iNOS (inducible nitric oxide synthase) was evaluated. Considering the endocytosis results, there is interplay between the concentration and efficiency of glyconanoparticles due to the larger endocytosis degree observed for mixed-shell glyconanoparticles. Thus, the experiment was performed under a similar degree of endocytosis. (The standard curves of endocytosis efficiency vs concentration of glyconanoparticles are presented in Figure S23, Supporting Information.) As shown in Figure 6, all glyconanoparticles stimulated higher expression of the
inflammatory cytokines compared to the control group. It was interesting that glyconanoparticles BP-MG and CP-MG promoted a higher release of arginase 1 and iNOS than BP-M/ G and CP-M/G. These results indicated that the MG glyconanoparticles are more efficient in macrophage activation than the M/G glyconanoparticles even when normalized to have the same number of particles uptaken by the cell, which is consistent with the stronger lectin binding and superior endocytosis efficiency of MG glyconanoparticles. The Corona Architectural Effect Comes from Phase Separation of Glycopolyesters. It is not hard to understand that the mixed-shell glyconanoparticles M/G and MG have a higher endocytosis efficiency than the corresponding nanoparticles with only one kind of sugar on the surface. MG and M/G have two kinds of saccharides, which recognize more types of receptors on macrophages than the single-sugar-shell particles. This obviously caused more efficient endocytosis. However, why do the blend-mixed-shell glyconanoparticles M/ G interact more weakly with receptors on macrophages and lectins than the MG from glycopolymers? Since both MG and M/G particles have similar particle sizes and ratios of Man to Gal, the differences between MG and M/G might be attributed to the saccharide distribution, i.e., corona architecture of the nanoparticles. It is well-known that when the micelle corona is made of two types of polymer chains, phase separation might happen;25 i.e., the unlike polymer chains tend to aggregate separately, forming individual domains. A simple model shown in Scheme 3 illustrates the difference between MG and M/G in interactions with cells. As shown in Scheme 3A, for MG, due to Man and Gal being molecularly mixed, they are available to interact with both the receptors for Man and Gal on the cell surface, leading to a high efficiency in cell interactions. However, for the case of M/G shown in Scheme 3B, where the cell surface contacts the phase of Gal-containing polymers, only the receptor CD301 for Gal plays its role while the receptor CD206 does not, because it could not reach the Man units. The opposite case of M/G is shown in Scheme 3C where the cell contacts the Man-containing phase, so only the interaction of Man−CD206 takes place. Phase separation Was Supported by the Crystallization Ability of Glycopolyesters. The high similarity between Gal and Man makes it almost impossible to examine their phase separation by an electronic microscope. Aliphatic polyesters PCL are well-known crystallizable polymers, and their crystallization behavior has been extensively studied. However, no work dealing with the effect of sugar pendants on the crystallization of polyesters was found in the literature. To investigate some clues of the phase separation in M/G, the crystallization behavior of glycopolyesters was studied. Solutions of P-BP-Man, P-BP-Gal, P-CP-Man, and P-CP-Gal and their polyester precursors BP and CP in DMF (20 mg/ mL) were evaporated and then observed by a polarizing microscope (Figure 7). We found that BP and P-BP-Man showed bright spots that significantly differed from the total dark field given by P-BP-Gal. The result indicated that the attachment of Man to BP did not alter its crystallization but that of Gal did. The TEM images (Figure S21, Supporting Information) support these results; i.e., Man did not affect BP crystallization, while the modification of Gal inhibited the crystallization, resulting in an amorphous polymer film. Meanwhile, it was also found that CP and P-CPMan had bright spots under the polarizing microscope (Figure S22A−C, Supporting Information). These phenomena were
Figure 6. Cytokine release under equal endocytosis amounts. Data are shown as the mean ± SEM of three independent experiments. Key: *, p < 0.05; **, p < 0.01. 14689
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Scheme 3. Illustration of the Endocytosis of Glyconanoparticles by Macrophages Proposed in This Study: (A) MG Type, (B, C) Two Possibilities of the M/G Type
confirmed by TEM. As shown in Figure S22D, P-CP-Man showed massive and clear regular structures with high contrast under TEM, which means that P-CP-Man was crystallized. Meanwhile, CP itself showed clear crystallization structures under TEM (Figure S22F). However, P-CP-Gal only showed irregular structures (Figure S22E). Clearly, the significant difference in crystallization ability between BP/CP-Man and BP/CP-Gal promotes the phase separation in the blend corona of M/G. It is worth mentioning that although reports on glycopolymers are easily found in the literature, no reports discussed the phase separation behavior of different glycopolymers.
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CONCLUSIONS In this study, a series of glyconanoparticles with various architectures have been designed and prepared. Briefly, mixedshell glyconanoparticles prepared by blending two homoglycopolymers, i.e., BP-M/G, CP-M/G, or from glycopolymers containing both Man and Gal, i.e., BP-MG and CP-MG, were carefully evaluated for their efficiency in macrophage endocytosis and lectin binding, under the condition of very similar particle sizes and the same Man/Gal ratio. The results showed that the MG glyconanoparticles formed by the glycopolymers gave much higher efficiency in cell and lectin interaction than the M/G particles formed by blending two homoglycopolymers. This “sweet” architecture effect has never been reported in the literature and could be attributed to the phase separation of two homoglycopolymers on the M/G particle surface, because the crystallizable BP/CP-Man was not compatible with the amorphous BP/CP-Gal.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.7b07768. Experimental details, characterization of the monomer and polyesters, cell viability test, preparation of fluorescein-loaded glyconanoparticles, confocal fluorescence microscopy images, inhibition test in vitro, aggregation test of glyconanoparticles, characterization of saccharide-modified polyesters, measurement of
Figure 7. Polarizing microscope images of (A) P-BP-Man, (B) P-BPGal, and (C) BP (scale bars 5 μm). 14690
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endocytosis standard curves, and degradation test of CPMG (PDF)
AUTHOR INFORMATION
Corresponding Author
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[email protected] ORCID
Guosong Chen: 0000-0001-7089-911X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We acknowledge the financial support from the National Natural Science Foundation of China (Grant Nos. 51721002, 21504016, and 91527305). We sincerely thank Mr. Brian Michael Seifried from Prof. Bradley Olsen’s MIT laboratory for his kind help with the manuscript revision. We also thank the Joint Lab for Structural Research at the Integrative Research Institute for the Sciences (IRIS Adlershof).
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DOI: 10.1021/jacs.7b07768 J. Am. Chem. Soc. 2017, 139, 14684−14692
