Letter pubs.acs.org/macroletters
Fluorescent Glyco Single-Chain Nanoparticle-Decorated Nanodiamonds Kilian N. R. Wuest,†,‡ Hongxu Lu,‡ Donald S. Thomas,§ Anja S. Goldmann,†,∥ Martina H. Stenzel,*,‡ and Christopher Barner-Kowollik*,†,∥ †
Macromolecular Architectures, Institut für Technische Chemie und Polymerchemie, Karlsruhe Institute of Technology (KIT), Engesserstr. 18, 76128 Karlsruhe, Germany ‡ Centre for Advanced Macromolecular Design (CAMD), University of New South Wales (UNSW), Sydney, NSW 2052, Australia § Mark Wainwright Analytical Centre, University of New South Wales (UNSW), Sydney, NSW 2052, Australia ∥ School of Chemistry, Physics and Mechanical Engineering, Queensland University of Technology (QUT), 2 George Street, Brisbane, QLD 4000, Australia S Supporting Information *
ABSTRACT: We introduce the light-induced collapse of single glycopolymer chains in water generating fluorescent glyco singlechain nanoparticles (SCNPs) and their subsequent functionalization onto nanodiamonds. The glycopolymer precursors are prepared by polymerizing an acetylated mannose-based methacrylate monomer followed by a deprotection and postpolymerization functionalization step, introducing profluorescent photoactive tetrazole groups and furan-protected maleimide moieties. Subsequent UV irradiation in highly diluted aqueous solution triggers intramolecular tetrazole-mediated cycloadditions, yielding glyco SCNPs featuring fluorescence as well as lectin binding properties. The obtained SCNPs are coated onto nanodiamonds by adsorption, and the obtained hybrid nanoparticles are in depth characterized in terms of size, functionality, and bioactivity. Different coating densities are achieved by altering the SCNP concentration. The prepared nanoparticles are nontoxic in mouse RAW 264.7 macrophages. Furthermore, the fluorescence of the SCNPs can be exploited to image the SCNP-coated nanodiamonds in macrophage cells via confocal fluorescence microscopy.
C
functionalities and properties. Different cross-linking approaches, such as photochemical ligations,20−23 thermal cycloadditions,24,25 and noncovalent interactions,26,27 have been employed for single-chain collapse. The light-induced profluorescent nitrile-imine-mediated tetrazole ene cycloaddition (NITEC) reaction has been exploited as an efficient cross-linking chemistry to prepare fluorescent SCNPs.20,28,29 The NITEC conjugation is based on 2,5-diphenyltetrazole groups. Upon UV irradiation, nitrogen is released, and a reactive nitrile imine structure is formed that undergoes 1,3-dipolar cycloadditions with suitable double bonds.30 The first example of NITEC SCNPs was reported in 2014 highlighting the fluorescence properties as well as the cross-linking chemistry.20 The pyrazoline cross-links formed during the NITEC reaction are highly fluorescent with a suitable excitation and emission wavelength for bioimaging applications. Subsequently, NITEC chemistry has been employed for the preparation of degradable SCNPs based on
arbohydrates play an important role in many biological processes, such as inflammation, cancer cell metastasis, and signal transmission.1 Mimicking carbohydrate structures with synthetic glycopolymers is a powerful approach to exploit their bioactivity for therapeutic applications.2 For instance, glycopolymers are recognized by cell surface receptors enabling active targeting for drug delivery applications.3 A variety of glycopolymer architectures have been investigated as bioactive compounds including dendrimers,4 star polymers,5,6 and micelles7−12 as well as glycopolymer-coated inorganic nanoparticles.13−15 A relatively new class of polymeric nanoparticlesso-called single-chain nanoparticles (SCNPs) have recently gained increasing interest in the nanoscience community but has not yet been applied in the context of synthetic glycopolymers.16−18 A SCNP is a single polymer chain that is intramolecularly cross-linked. Very small nanoparticles can be prepared with sizes not accessible by conventional nanoparticle synthesis approaches such as emulsion polymerization or nanoprecipitation.19 Depending on the molecular weight of the polymeric precursor and the cross-linking density, nanoparticle diameters below 10 nm are readily accessible. Moreover, the straightforward bottom up approach allows for the design of nanoparticles with variable © XXXX American Chemical Society
Received: August 26, 2017 Accepted: September 28, 2017
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DOI: 10.1021/acsmacrolett.7b00659 ACS Macro Lett. 2017, 6, 1168−1174
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ACS Macro Letters metathesis polymers28 as well as water-soluble SCNPs based on poly(acrylic acid).29 Due to their small sizes, SCNPs represent an interesting class of nanomaterial for the structuring of surfaces. In nature, nanostructured surfaces are omnipresent. For instance, the surface of many viruses is decorated with glycoprotein spikes, which interact with cell receptors to induce targeted cellular uptake.31−34 Different approaches to mimic viral surface structures on synthetic particles have been reported. For example, triblock terpolymers have been self-assembled into nanoparticles with virus-like morphologies and efficient cellular uptake in macrophages.35 Furthermore, silica nanoparticles (∼200 nm) were decorated with smaller silica nanoparticles (∼6 nm) to generate a surface topography inspired by viruses, resulting in increased cellular uptake.36,37 The use of polymeric SCNPs instead of inorganic nanoparticles for surface structuring represents an elegant alternative since the soft nature of SCNPs resembles natural structural elements more accurately. Herein, the preparation of glycopolymeric SCNPs and their adsorption onto nanodiamonds (NDs) is introduced. The cross-linking chemistry for the formation of SCNPs is based on aqueous light-triggered NITEC chemistry. Absolute size distribution information was extracted from size exclusion chromatography (SEC) data. The obtained SCNPs are fluorescent, water-soluble, and bioactive. Inspired from glycoprotein spikes on viruses, the glyco SCNPs were used to decorate the surface of NDs. We demonstrate that the bioactivity of the SCNPs is transferred to the hybrid particles. Furthermore, the cytotoxicity as well as confocal fluorescence imaging in macrophages are explored. The described approach to glyco SCNPs and glyco SCNP decorated NDs thus provides a novel synthetic platform in the realm of glyco-nanotechnology for viral mimics. Initially, an acetyl-protected mannose-based methacrylate monomer was polymerized via reversible addition−fragmentation chain transfer (RAFT) polymerization (Scheme 1). Mannose glycopolymers were prepared since the overexpressed mannose receptors on macrophages represent an interesting target for therapeutic applications. 38 Two well-defined glycopolymers with low dispersities and different chain lengths (Mn = 18100 g mol−1 and Mn = 35800 g mol−1; Đ = 1.12, Figure S1) were prepared. The deprotection of the hydroxyl groups was performed with sodium methylate in the crude polymerization mixture directly after the polymerization. The complete absence of any acetyl proton signals in the 1H NMR spectra of the polymers indicates full deprotection (Figures S5−S6). Further, the deprotection induced the cleavage of the dithiobenzoate end groups. Since the deprotection was performed in the crude polymerization mixture, unreacted monomer was present, which probably capped the thiol end groups with a sugar unit in a thiol-Michael addition. The crosslinking groups, i.e., the photoactive tetrazole moieties (Tet) and furan-protected maleimide groups (pMal), were introduced by a simple one-pot esterification reaction of the mannose hydroxyl groups (Scheme 1). The pendant mannose groups have three secondary and one primary hydroxyl group, where the functionalization can occur. Likely the functionalization takes place preferentially at the primary hydroxyl group due to its highest reactivity.39 The functionalization results are summarized in Table 1. A functionalization degree (relative amount of functional groups per repeating unit) of 8% Tet and 10% pMal was achieved, as observed by 1H NMR spectroscopy.
Scheme 1. Synthetic Approach for the Preparation of SCNPDecorated Nanodiamondsa
a
The polymeric precursors were prepared by RAFT polymerization and subsequent esterification of mannose hydroxyl groups. Lightinduced single-chain cross-linking in water yield SCNPs, which were immobilized onto nanodiamonds by adsorption in water.
Moreover, a polymer with a higher Tet content (20%) was prepared in order to investigate the effect of cross-linking density on SCNP properties. The light-induced intramolecular compaction reactions were performed in highly diluted aqueous solutions (16.7 mg L−1) to prevent intermolecular reactions. A commercially available light source (λmax = 320 nm) was used to trigger the cross-linking reactions. Size exclusion chromatography was employed to monitor the single chain collapse. The light-induced crosslinking leads to an increase in retention time (Figures S2−4) indicating a decrease in hydrodynamic volume. The success of SCNP formation is typically evidenced by a change in SEC retention time since intramolecularly cross-linked polymer chains possess a smaller hydrodynamic volume than their noncross-linked precursors. However, a change in retention time, or the frequently reported change in apparent molecular weight, does not provide any absolute size information. The hydrodynamic volume SEC analysis was introduced for starch analysis by Gilbert and co-workers.40 Herein, we describe a similar approach for the characterization of SCNPs. It is based on the separation of SEC by hydrodynamic volume41 and its calibration with standards of known molecular weights that are converted into corresponding hydrodynamic diameters. The correlation of the molecular weight (M) and hydrodynamic volume (Vh) of a monodisperse polymer is given by the following equation ([η]: intrinsic viscosity, Na: Avogadro constant):40 Vh =
2[η]M 5Na
(1)
The Mark−Houwink-Sakurada (MHS) equation describes the empirical correlation between the molecular weight M and the intrinsic viscosity [η]: [η] = KM α 1169
(2) DOI: 10.1021/acsmacrolett.7b00659 ACS Macro Lett. 2017, 6, 1168−1174
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ACS Macro Letters Table 1. Characterization Data of SCNP Precursors P1−P3a
a
polymer
Mn,NMR/g mol−1
DPNMR
Mn,SEC/g mol−1
Đ
% Tet
% pMal
P1 P2 P3
32000 61000 66500
94 180 180
24400 45000 51500
1.20 1.18 1.25
8 8 20
10 10 8
The relative (compared to repeating units) amount of Tet and pMal (% Tet and % pMal) were calculated using 1H NMR spectroscopy.
The MHS parameters K and α are determined empirically and are known for common calibration standards and typical SEC operation conditions (i.e., solvent/temperature combinations). The combination of eqs 1 and 2 allows for the calculation of absolute SEC size distributions from SEC molecular weight plots (volume was transformed into a diameter): Dh = 2· 3
3KM α+ 1 10πNa
studies,20 increasing the tetrazole content in the precursor polymer (P3 compared to P2) leads to a higher compaction of the polymer chains (35% compared to 19%) due to more crosslinks. Furthermore, diffusion-ordered NMR spectroscopy (DOSY) in D2O was employed as a complementary method to SEC to obtain relative size information about the single-chain collapse (Figure S11). In all three systems, an increase of the diffusion coefficient (DDOSY diff ), correlating with a decrease in hydrodynamic volume, was observed, confirming the successful formation of SCNPs (Figure 1). It should be noted that the diffusion coefficient describes the systems only partially since distribution is challenging to information about the DDOSY diff access, and thus for its calculation a monodisperse system was assumed. As shown in previous studies, the irradiation of tetrazole functional polymers in the absence of any double bonds leads to cross-linking reactions,20 which are probably based on the formation of tetrazines from nitrile imine dimerization.42 The cross-linking chemistry of Tet/pMal-based SCNPs is based on a combination of NITEC reactions and nitrile imine dimerization, which form pyrazoline and tetrazine cross-links, respectively (Scheme 1).20 Structural information regarding the SCNP formation was obtained from 1H NMR spectroscopy (Figures S7−9). The decrease of the olefin proton signals upon single-chain collapse as well as the appearance of aromatic proton signals at 8.04, 7.91, 7.18, and 6.94 ppm corresponding to the cycloadducts confirm the success of the cross-linking reactions.29 Furthermore, the conversion of the pMal groups can be calculated from the decrease of the olefin resonance at 6.55 ppm. Whereas 42% and 63% of the pMal groups reacted in P1 and P2, respectively, full conversion of the pMal groups in P3 was observed due to a higher Tet concentration in the precursor. UV−vis and fluorescence spectra of the prepared SCNPs were recorded (Figure S10). All SCNPs have an absorbance band with a maximum at 412 nm and possess a strong fluorescence emission band at λmax = 555 nm corresponding to the pyrazoline cycloadducts from the NITEC reaction. The fluorescence properties are suitable for imaging applications as demonstrated below. Next, a qualitative Concanavalin A (ConA) turbidity assay was performed to evaluate the bioactivity of the SCNPs.43 ConA is a lectin that exists as a tetramer. The binding of pendant mannose units in glycopolymers to ConA leads to an aggregation-induced increase in turbidity, which can be monitored by UV−vis spectroscopy. Upon mixing of the prepared glyco SCNPs with ConA, an increase in turbidity over time was observed (Figure S12) showing the successful lectin binding of each SCNP sample. The binding to ConA demonstrates the ability of the glyco SCNPs to act as targeting structures for biomedical applications. Nanoparticles have been coated with glycopolymers to enhance colloidal stability and improve therapeutic performance.3 Nanodiamonds (NDs) are carbon nanoparticles with
(3)
In the current study, the SEC measurements were performed in DMF (0.1 M LiBr) at 50 °C, and the system was calibrated with PMMA standards. Using the MHS parameters of PMMA and eq 3, the molecular weight distributions of the measured samples could be transformed into Dh distributions. The calibration method is independent of polymer type and structure. According to SEC, the diameter of the precursors P1−P3 decreased significantly upon UV irradiation evidencing the formation of sub 10 nm SCNPs (Figure 1). The reduction in diameter for the larger polymers (P2 and P3) is more distinct (19% and 35%) compared to P1 (7%) due to the presence of more cross-links per chain. In agreement with previous
Figure 1. SEC determined diameter plots of precursors P1−P3 and SCNP1−SCNP3 in DMF. Average hydrodynamic diameters (DSEC h ) as DOSY ) determined from DOSY well as diffusion coefficients (Ddiff measurements are displayed. 1170
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ACS Macro Letters
Figure 2. Characterization of SCNP ND hybrids. (A) Adsorption assay of SCNP2 to NDs. The data points were fitted to a Langmuir−Freundlich isotherm. (B) Thermogravimetric analysis (red: ND precursor; green: SCNP2; black: ND@SCNP-9%). (C) DLS hydrodynamic size distributions (solid lines: intensity average, dashed lines: number-average) of NDs (red) and ND@SCNP-9% (black). (D) DLS ConA assay (z-average diameters) of SCNP-coated NDs (black: ND@SCNP-3%, green: ND@SCNP-6%, blue: ND@SCNP-9%) and of noncoated NDs (red) as a control. (E) TEM images of ND@SCNP-9% before (left) and after the addition of ConA (right).
Table 2. Characterization Data of Prepared NDsa sample
grafting density/wt %
ND precursor ND@SCNP-3% ND@SCNP-6% ND@SCNP-9%
2.8 6.0 9.3
SCNP footprint/nm2
surface coverage/%
−
−
105 47 30
28 62 100
Dh/nm (number)
Dh/nm (intensity)
PDI
57 68 72 72
97 102 111 119
0.108 0.071 0.083 0.102
a
Hydrodynamic diameters (Dh) were obtained from DLS measurements in water. SCNP footprints and surface coverages were estimated using the Dh value of the ND precursor, density of diamond, and molecular weight information of SCNPs.
high potential for biomedical applications. 44 They are commercially available in large quantities, biocompatible, and possess interesting fluorescence properties. Polymers have been grafted from45,46 and to NDs,47,48 but the simplest approach to coat NDs with polymers is by surface adsorption.49 Herein, a simple and straightforward approach to obtain SCNP-coated NDs with controlled grafting densities is introduced. First, the surface of high-pressure high-temperature (HPHT) NDs was oxidized by an acid mixture (H2SO4 (96%) / HNO3 (67%), 9/ 1, v/v) treatment prior to the coating process. Although not studied in the current contribution, hydrogen bonds are proposed to play an important role for the attachment of glyco SCNPs to oxidized NDs. An extensive IR study by Holt and co-workers supports the idea that hydroxyl groups form hydrogen bonds with oxidized nanodiamonds.50 To investigate the adsorption of SCNPs onto NDs, a simple adsorption assay with SCNP2 was performed. The amount of adsorbed polymer increased with increasing SCNP concentration and leveled out at 11 wt % (Figure 2A). Thermogravimetric analysis of the SCNP-coated NDs after several washing cycles with water revealed a grafting density of 9.3 wt %, showing a high coating stability in water (Figure 2B, Table 2). The surface coverage was estimated using the density of diamond, the hydrodynamic diameters of nanoparticles, and the molecular weight of
SCNP2. A grafting density of 9.3 wt % corresponds to a SCNP footprint (surface area per SCNP) of 30 nm 2 corresponding to a circle of a diameter of 6.1 nm, which is slightly smaller than the hydrodynamic diameter of the SCNPs. The minimal difference in polymer footprint and hydrodynamic size of SCNP2 as well as the fact that increasing SCNP concentration does not lead to higher adsorption densities indicates that the ND surface is saturated with SCNPs. Dynamic light-scattering (DLS) experiments revealed that upon SCNP adsorption the hydrodynamic diameter increases from 57 to 72 nm (number-average value, Figure 2C, Table 2) corresponding to a 7.5 nm thick surface layer. Inspired from the adsorption assay (Figure 2A) patchy surface structures were created on the surface of NDs by lowering the SCNP concentration in the adsorption experiment to 0.1 and 0.2 mg mL−1. The reduction of the SCNP concentration lead to glyco SCNP/ND hybrid particles with a grafting density of 2.8 and 6.0 wt % (determined by TGA, Figure S14) corresponding to an estimated surface coverage of 28% and 62%, respectively (Table 2). In DLS experiments, an increase in hydrodynamic diameter with increasing amounts of SCNPs on the ND surface was observed (Figures S15 and Table 2). 1171
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nanoparticles were successfully internalized by the cells and mainly distributed in the cytosol. Thus, glyco SCNPs constitute a well-functioning building block for the fabrication of fluorescent and bioactive nanoparticles. In summary, we pioneer fluorescent single-chain nanoparticles via the tetrazole-mediated light-triggered cross-linking of single mannose-based glycopolymer chains in water and subsequently adsorbed these onto nanodiamonds. Concomitantly, we introduce a SEC diameter analysis method for the fast and facile size distribution analysis of SCNPs. We submit that SEC diameter plots are a meaningful way of presenting SEC data for SCNPs. Upon UV irradiation, a significant decrease in hydrodynamic diameter as a consequence of intramolecular cross-linking was observed. The prepared glyco SCNPs possess fluorescence properties suitable for bioimaging applications. Furthermore, glyco SCNPs bind to ConA as shown in a turbidity assay. Subsequently, the glyco SCNPs were immobilized on nanodiamonds, and the bioactivity of the obtained hybrid particles was demonstrated in a ConA DLS aggregation assay. Variable amounts of SCNPs (2.8, 6.0, and 9.3 wt %) were coated onto NDs to obtain patchy surface structures. The SCNPs as well as the SCNP-coated NDs are nontoxic. The fluorescence of the SCNPs can be exploited to localize the SCNP-modified NDs in cells using confocal fluorescence microscopy techniques. The presented approach thus critically expands the field of glycopolymer nanoparticles with a very small, size tunable, fluorescent, and bioactive nanoobject, whose full potential for biological applications is currently under investigation.
Importantly, the bioactivity of the SCNPs was directly transferred to the ND hybrid particles. In the presence of ConA, the SCNP-coated NDs aggregate due to the binding of the ND surface-expressed mannose units to ConA. The aggregation was monitored online via DLS. The hydrodynamic diameter of glyco SCNP-coated NDs increased significantly, whereas noncoated NDs (control sample) showed no aggregation in the presence of ConA (Figure 2D). A lower aggregation rate was observed for ND@SCNP-3% (Table 2) due to less mannose SCNPs on the NDs. In agreement with the DLS assay, transmission electron microscopy (TEM) confirms the formation of large aggregates in the presence of ConA (Figure 2E). Mannose functional nanoparticles are powerful entities for targeting macrophages since mannose receptors are highly expressed on macrophage cells and allow receptor-mediated endocytosis.38 Thus, the cytotoxicity in mouse RAW 264.7 macrophages was investigated. It was shown that all nanoparticles are nontoxic at a concentration of 100 μg mL−1, which is important for any biological application (Figure 3A). In the
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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/acsmacrolett.7b00659. Experimental section, 1H NMR spectra, UV−vis/ fluorescence spectra, ConA turbidity assay for SCNPs, DLS plots, DOSY data (PDF)
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AUTHOR INFORMATION
Corresponding Authors
*E-mail:
[email protected] (C.B.-K.),
[email protected]. *E-mail:
[email protected] (M.H.S.).
Figure 3. (A) Cytotoxicity study of SCNPs (SCNP2) and SCNPfunctionalized NDs at a concentration of 0.1 mg mL−1 on mouse RAW 264.7 macrophages. (B) Confocal fluorescence images of macrophages incubated with nanoparticles for 1 h. Left: fluorescence images (excitation at 488 nm, emission at 493−634 nm). Center: differential interference contrast (DIC) image. Right: merged image. Scale bar = 20 μm.
ORCID
Anja S. Goldmann: 0000-0002-1597-2836 Martina H. Stenzel: 0000-0002-6433-4419 Christopher Barner-Kowollik: 0000-0002-6745-0570 Author Contributions
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
context of the current study, we demonstrate that this concentration will not cause any toxic effect in the cell uptake studies. The SCNPs introduced lectin-binding mannose units as well as fluorescent pyrazoline groups to the NDs. It should be noted that the NDs used in the current study are not fluorescent. High-energy irradiation followed by thermal annealing is usually required to induce the ND’s fluorescence51,52 but was not necessary for the herein investigated particles due to the fluorescence of the SCNPs. As a proof of concept, confocal fluorescence microscopy was conducted, allowing the localization of the modified NDs in macrophage cells (Figure 3B). The fluorescence images indicate that the
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS A.G., M.S., and C.B.-K. acknowledge the German Research Council (DFG) for funding (project BA3751/34-1). C.B.-K. acknowledges continued support from the Karlsruhe Institute of Technology (KIT) via the Helmholtz BioInterfaces in Technology and Medicine (BIFTM) as well as the Science and 1172
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Technology for Nanosystems (STN) program. C.B.-K. acknowledges key support from the Queensland University of Technology (QUT) as well as the Australian Research Council (ARC) in the form of a Laureate Fellowship.
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