Synthesis and characterisation of uniform CoPt nanoparticles using red blood cell ghosts conjugated with peptides on their inner surface

Abstract

We have demonstrated the potential of red blood cell ghosts (RBCGs) conjugated with a cobalt platinum (CoPt)-binding peptide as a scaffold for synthesising CoPt nanoparticles (NPs). The results of characterisation experiments showed that the NPs prepared using peptide-conjugated RBCGs were larger, more uniform, and had a higher Co content and improved magnetic properties.

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Hybrids of metal nanoparticles (NPs) and lipid vesicles are attractive materials in various fields, including catalysis, sensing, and biomedicine.^1–6 Lipid vesicles can entrap metal NPs on their surfaces, in the lipid bilayer, or within their aqueous cores, resulting in the stabilisation of metal NPs in aqueous environments. Metal NP–lipid vesicle hybrids have been prepared using several methods, such as lipid film hydration in the presence of colloidal metal NPs and in situ synthesis of metal NPs within lipid vesicles.^1,3,5 Although the hydration method is simple and versatile, it generally suffers from low yield, poor selectivity, and low purity because of the difficulty in controlling the location of metal NPs in lipid vesicles and separating free metal NPs and lipid vesicles. Direct synthesis within the confined nanospace of lipid vesicles enables the synthesis of metal NPs under mild conditions and allows the control of their size, composition, and morphology. However, the precise control of the formation process of metal NPs remains difficult.

Red blood cell ghosts (RBCGs), which are micro-sized biological membranes, are suitable scaffolds for the simultaneous synthesis and immobilisation of metal NPs. RBCGs are red blood cells (RBCs) from which cytoplasmic components are removed by hypotonic haemolysis.^7,8 Notable structural features of RBCGs include that they are biconcave hollow vesicles with large volume and surface area; in addition, they possess a unique two-dimensional triangular network called the “cytoskeleton” beneath their inner membrane.^9,10 The cytoskeleton, which is composed of proteins, enables the immobilisation of various functional molecules on the internal surface by the chemical modification of amino acids.^11,12 RBCGs with these structural features can be used as a space for controlling the synthesis and reactivity of inorganic materials. However, RBCGs have mainly been used as carriers for DDSs because of their high biocompatibility and drug-loading capacity.^13–15 The development of hybrids of RBCGs and inorganic materials remains challenging.

In this paper, we describe the design of RBCGs for the synthesis of bimetallic cobalt–platinum nanoparticles (CoPt NPs) with diverse functions such as excellent magnetic properties and catalytic activities.^16–18 In general, the physical properties of CoPt NPs depend on factors such as the particle size, shape, crystalline nature, and Co/Pt ratio.^19–21 To deposit uniform CoPt NPs on the inner surface of RBCGs, we fabricated RBCGs in which a CoPt-binding peptide was chemically attached to the RBCG cytoskeleton (Fig. 1). CoPt NPs were synthesised using peptide-conjugated RBGCs or RBCGs with various concentrations of CoCl₂ and K₂PtCl₄. The resulting CoPt NPs were characterised using metal quantification, TEM, and magnetic measurements.

Fig. 1 Schematic representation of the preparation of CoPt@RBCG_Co1P10pep.

We selected a CoPt-binding peptide (HYPTLPLGSSTY) (Co1P10) identified from a phage peptide display library to control the nucleation and growth of CoPt NPs on the interior surface of RBCGs.^22,23 R. R. Naik et al. reported that the synthesis of CoPt NPs using Co1P10 as a template produced stable and discrete crystalline CoPt NPs.²³ Chemical modification of Co1P10 onto the RBCG cytoskeleton was performed as described in our previous study (Fig. 1).¹¹ Briefly, the maleimide group was covalently conjugated to lysine residues of RBCG membrane proteins using a cross-linker ((4-maleimidobutyryloxy)sulfosuccinimide (sulfo-GMBS)), and then the resulting RBCG-bearing maleimide group was reacted with the cys-Co1P10 peptide (CGF_CNGHYPTLPLGSSTY) containing a maleimide-reactive cysteine residue introduced at the N-terminus of Co1P10 to obtain RBCG_Co1P10pep. The phase-contrast microscopy images of RBCG_Co1P10pep and RBCGs (Fig. S1†) show that RBCG_Co1P10pep had an average size of approximately 5 μm without membrane disruption owing to peptide modification. The cys-Co1P10 peptide contained F_CN, a non-natural phenylalanine derivative with a cyano group, and modification of the peptide onto RBCGs was confirmed by Fourier transform infrared (FTIR) spectroscopy. The FTIR spectrum of RBCG_Co1P10pep showed a characteristic peak of the cyano group at 2229 cm⁻¹, which was not observed in the FTIR spectrum of RBCGs (Fig. 2a). Furthermore, the FTIR spectrum of the control sample, prepared by reacting RBCGs (without maleimide groups) with the cys-Co1P10 peptide, did not show a peak corresponding to the cyano group. These results indicated that the Co1P10 peptide was modified onto RBCGs via a thioether bond between maleimide and cysteine (Fig. 1).

Fig. 2 (a) FTIR spectra of RBCG_Co1P10pep, RBCGs, and the control sample (RBCG + cys-Co1P10 peptide). (b) Phase-contrast and fluorescence microscopy images of RBCG_{Co1P10-Flupep}.

A fluorescein-labelled Co1P10 peptide (cys-Co1P10-Flu peptide, Ac-CGF_CNGHYPTLPLGSSTYK(Flu)-COOH) was used to confirm the modification site of the peptide on RBCGs and the approximate number of peptides modified per RBCG (N_pep). Fluorescence microscopy images of RBCG_{Co1P10-Flupep} revealed fluorescein-derived fluorescence near the RBCG membrane (Fig. 2b). The SDS-PAGE of RBCG_{Co1P10-Flupep} (Fig. S2†) showed that the fluorescein-derived fluorescence was observed from the bands of α-spectrin, β-spectrin and band 3, revealing that the peptides are covalently conjugated to cytoskeletal proteins. The UV–vis spectrum of RBCG_{Co1P10-Flupep} treated with 1% SDS showed an absorption peak at 493 nm derived from fluorescein (Fig. S3†), and the N_pep value estimated from its absorbance was 2.2 × 10⁶ (see the ESI† for calculation details).

The in situ synthesis of CoPt NPs was performed using RBCG_Co1P10pep or RBCGs. RBCG_Co1P10pep or RBCGs (protein concentration of 192 μg mL⁻¹) were pre-incubated in buffer with varying concentrations of CoCl₂ and K₂PtCl₄ and then reduced with NaBH₄ (10 equivalents to the total metal concentration). The reaction mixture immediately became dark brown owing to the formation of CoPt NPs. CoPt@RBCG_Co1P10pep or CoPt@RBCG was purified by centrifugation. For example, Co_0.20Pt_0.20@RBCG, Co_0.30Pt_0.30@RBCG, and Co_0.40Pt_0.40@RBCG were prepared with CoCl₂/K₂PtCl₄ concentrations of 0.20/0.20, 0.30/0.30, and 0.40/0.40 mM, respectively. The subscripts for Co and Pt in the composite names represent the CoCl₂ and K₂PtCl₄ concentrations at pre-incubation, respectively. Phase-contrast microscopy images of Co_0.20Pt_0.20@RBCG, Co_0.30Pt_0.30@RBCG, and Co_0.40Pt_0.40@RBCG (Fig. S4†) showed that only Co_0.40Pt_0.40@RBCG exhibited aggregation and significant shape changes or membrane disruption. Therefore, the total metal concentration was set to 0.40 or 0.60 mM at pre-incubation, and the CoCl₂/K₂PtCl₄ concentration ratio was changed for the synthesis (Fig. 3). The Co and Pt concentrations in the CoPt@RBCG_Co1P10pep and CoPt@RBCG suspensions were determined using ICP-OES. Fig. 3 shows the Co and Pt concentrations per 100 μg mL⁻¹ protein concentration (C_Co and C_Pt). The value shown on each bar is C_Co/C_Pt, which is the composition of the resulting nanoparticles as determined by ICP-OES analysis. We found the following: in both CoPt@RBCG_Co1P10pep and CoPt@RBCG samples, (1) the C_Total (C_Co + C_Pt) values of the samples prepared at the total metal concentration of 0.60 mM are higher than those of the samples prepared at 0.40 mM, (2) the C_Co values increase as the CoCl₂ concentration increases, and (3) the C_Co value of CoPt@RBCG_Co1P10pep was higher than that of CoPt@RBCG at each CoCl₂/K₂PtCl₄ concentration, resulting in a higher C_Co/C_Pt ratio. For example, Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG had C_Co/C_Pt ratios of 5.3 and 3.0, respectively. Thus, the modification of the Co1P10 peptide onto RBCGs enabled an increase in Co content. This result is reasonable because Co1P10 binds to cobalt nanoparticles identified in a phage peptide display library.²³

Fig. 3 Stacked bar graph summarizing Co and Pt concentrations (C_Co and C_Pt) per 100 μg mL⁻¹ protein concentration for CoPt@RBCG_Co1P10pep and CoPt@RBCG prepared with various concentrations of CoCl₂ and K₂PtCl₄. Each value is the average of at least three experimental repeats. Error bars represent the mean ± standard deviation of C_Co (blue) and C_Pt (red). Values shown on each bar represent the composition (C_Co/C_Pt) of the resulting nanoparticles as determined by ICP-OES analysis.

Further characterisation of Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG, which had high C_Co and C_Total values, was performed using phase-contrast microscopy and transmission electron microscopy (TEM). The phase-contrast microscopy images of Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG showed no aggregation, significant shape change, or membrane disruption (Fig. S5†). The TEM images of Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG at low magnification confirmed the spherical structure of RBCGs (Fig. 4a and b (left)). At high magnification (Fig. 4a and b (middle)), discrete spherical electron-dense NPs and clear crystalline lattices were observed on the RBCG membrane. However, in Co_0.45Pt_0.15@RBCG, NPs without a crystalline lattice are also observed, suggesting that their crystallinity is lower than that of the NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep. This indicates that the Co1P10 peptide on RBCGs influenced the crystalline nature of the NPs. The lattice spacing of the NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep is 0.21–0.22 nm (Fig. S6†), which is close to the 0.21 nm value observed in the synthesis of CoPt NPs using Co1P10 as a template reported by R. R. Naik et al.²³ Therefore, the NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep might be similar to the CoPt NPs synthesized by R. R. Naik et al. The energy-dispersive X-ray spectroscopy (EDS) point analysis of a NP in Co_0.45Pt_0.15@RBCG is shown in Fig. S7.† Although the Co Kα peak was confirmed in the EDS spectrum, it was difficult to identify the peaks derived from Pt (M and Lα). This was because the Pt M peak overlapped with the strong Kα peak of phosphorus derived from phospholipids, and the Pt Lα peak was not detected because of the detection limit caused by the low Pt content. From the size distribution histograms of CoPt NPs, the average diameters of the CoPt NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG were determined to be 1.43 ± 0.31 and 1.01 ± 0.72 nm, respectively (Fig. 4a and b (right)). It is noteworthy that Co_0.45Pt_0.15@RBCG_Co1P10pep exhibited a narrower particle size distribution due to a significant decrease in the particle population of CoPt NPs smaller than 1 nm. This narrower distribution can be attributed to a reduction in random nucleation. Therefore, it is believed that nucleation and growth sites are restricted by the Co1P10 peptide on the RBCG membrane. These results indicate that RBCG_Co1P10pep facilitates the formation of CoPt NPs with higher monodispersity and crystallinity than those prepared using RBCG alone.

Fig. 4 Low-magnification (left) and high-magnification (middle) TEM images and size distribution histograms of CoPt NPs (right) of (a) Co_0.45Pt_0.15@RBCG_Co1P10pep and (b) Co_0.45Pt_0.15@RBCG. The error indicates the standard deviation. (c) Field-dependent magnetisation curves of freeze-dried Co_0.45Pt_0.15@RBCG_Co1P10pep, Co_0.45Pt_0.15@RBCG and RBCGs at 5 K. The observed magnetisation was divided by the sample weight.

As control experiments, we performed the synthesis of M_0.60@RBCG_Co1P10pep and M_0.60@RBCG (M = Co or Pt), the synthesis of CoPt NPs in the presence and absence of Co1P10 without RBCGs, and the synthesis of CoPt NPs using RBCG_Co1P10pep with a reduced amount of the Co1P10 peptide on the inner surface of RBCGs. The TEM images of Co_0.60@RBCG_Co1P10pep, Co_0.60@RBCG, Pt_0.60@RBCG_Co1P10pep, and Pt_0.60@RBCG confirmed the formation of NPs in all samples (Fig. S8†). The C_Pt values of Pt_0.60@RBCG_Co1P10pep and Pt_0.60@RBCG were almost the same, but the C_Co value of Co_0.60@RBCG_Co1P10pep was higher than that of Co_0.60@RBCG (Fig. S8†). These results are consistent with the higher C_Co observed in CoPt@RBCG_Co1P10pep compared to CoPt@RBCG. The synthesis of NPs in the presence and absence of Co1P10 without RBCGs was performed at a Co1P10 concentration of 2.6 μM or 260 μM, with CoCl₂/K₂PtCl₄ concentration of 0.45/0.15 mM. The rationale for setting the Co1P10 concentration to 2.6 μM or 260 μM was that the peptide concentration determined from RBCG_{Co1P10-Flupep} was 2.6 μM per 192 μg mL⁻¹ of protein. The TEM image of NPs synthesized in the absence of both Co1P10 and RBCGs shows large aggregates of NPs (Fig. S9a†). TEM images of the CoPt NPs synthesized at Co1P10 concentrations of 2.6 μM and 260 μM show spherical electron-dense NPs with a crystalline lattice (Fig. S9b and c†). The average diameters of the CoPt NPs synthesized at Co1P10 concentrations of 2.6 μM and 260 μM were 1.96 ± 0.27 and 3.05 ± 0.78 nm, respectively (Fig. S9b and c†). The particle size of the CoPt NPs in Co_0.45Pt_0.15RBCG_Co1P10pep was smaller than that of the CoPt NPs synthesized at a Co1P10 concentration of 2.6 μM, suggesting that the confined environment of RBCGs surrounded by the membrane may affect the nucleation and growth of NPs. RBCG_Co1P10pep with a reduced amount of the Co1P10 peptide on the RBCG membrane (RBCG_{Co1P10pep(reduced)}) was prepared by decreasing the concentration of the cys-Co1P10 peptide from 0.30 mM to 0.15 mM in the preparation of RBCG_Co1P10pep. The average diameter of the CoPt NPs in Co_0.45Pt_0.15RBCG_{Co1P10pep(reduced)} was 1.32 ± 0.23 nm (Fig. S10†), which was slightly smaller than that of Co_0.45Pt_0.15@RBCG_Co1P10pep. This result is consistent with that obtained from the synthesis of CoPt NPs in the presence of Co1P10 without RBCGs, where the particle size increased with higher peptide concentration. The amount of the Co1P10 peptide modified on the RBCG membrane was found to be an important factor in controlling the particle size.

The magnetic properties of freeze-dried Co_0.45Pt_0.15@RBCG_Co1P10pep, Co_0.45Pt_0.15@RBCG and RBCGs were characterised using a superconducting quantum interference device (SQUID) magnetometer. RBCGs were diamagnetic. The field-dependent magnetisation curves of Co_0.45Pt_0.15@RBCG_Co1P10pep and Co_0.45Pt_0.15@RBCG at 5 K exhibited no hysteresis loops, indicating a paramagnetic behaviour. However, Co_0.45Pt_0.15@RBCG_Co1P10pep showed a larger increase in magnetisation and a clearer inflection point than Co_0.45Pt_0.15@RBCG, which may be due to the formation of a larger magnetic domain within the CoPt NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep. The magnetic properties of the CoPt NPs depend on the particle size and Co/Pt ratio; the magnetisation increases with increasing particle size and/or Co composition of the NPs.^19–21 Therefore, the observed magnetic behaviour is consistent with the fact that the CoPt NPs in Co_0.45Pt_0.15@RBCG_Co1P10pep have a larger particle size and higher Co content than those in Co_0.45Pt_0.15@RBCG.

Conclusions

We demonstrated a simple approach for dispersing and immobilising well-defined bimetallic CoPt NPs on the large inner surface of RBCGs. The synthesis of CoPt NPs using RBCGs with the Co1P10 peptide, which was covalently conjugated to cytoskeletal proteins, enabled the formation of larger and size-restricted CoPt NPs with higher crystallinity and Co content than those prepared using RBCGs without peptides. These features of CoPt NPs resulted in improved magnetic properties. RBCGs deposited with magnetic NPs might undergo shape deformation under an external magnetic field, potentially enabling the development of magnetically responsive materials. We believe that our method can be applied to the synthesis of a wide range of bimetallic NPs and inorganic materials, leading to the creation of diverse biomembrane–inorganic hybrid materials for biomedical applications.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

Part of this work (TEM) was supported by the “Advanced Research Infrastructure for Materials and Nanotechnology in Japan (ARIM)” of the Ministry of Education, Culture, Sports, Science and Technology (MEXT). Proposal Number: JPMXP1223NR0033. The authors thank the Industrial Research Center of Shiga Prefecture, Japan, for ICP-OES. Magnetic measurements were performed at the Analytical Instrument Facility of the Graduate School of Science, Osaka University. This work was supported by JSPS KAKENHI Grant Numbers 19H04599 and 22K05133 (to T. K.).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d5dt00502g

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