Laura Maria
Slavu‡
a,
Antonella
Antonelli‡
*b,
Emanuele Salvatore
Scarpa
b,
Pasant
Abdalla
b,
Claire
Wilhelm
c,
Niccolò
Silvestri
d,
Teresa
Pellegrino
d,
Konrad
Scheffler
e,
Mauro
Magnani
b,
Rosaria
Rinaldi
*afg and
Riccardo
Di Corato
*gh
aMathematics and Physics “E. De Giorgi” Department, University of Salento, Via Monteroni, Lecce 73100, Italy. E-mail: ross.rinaldi@unisalento.it
bDipartimento di Scienze Biomolecolari, Università degli Studi di Urbino Carlo Bo, Via Saffi 2, Urbino 61029, Italy. E-mail: antonella.antonelli@uniurb.it
cLaboratoire Physico Chimie Curie, UMR 168, CNRS, Institut Curie, PSL University, Sorbonne Université, 75005 Paris, France
dItalian Institute of Technology, via Morego 30, 16131 Genoa, Italy
eSection for Biomedical Imaging, University Medical Center Hamburg-Eppendorf, 22529 Hamburg, Germany
fScuola Superiore ISUFI, University of Salento, Via Monteroni, University Campus, Lecce 73100, Italy
gInstitute for Microelectronics and Microsystems (IMM), CNR, Via Monteroni, Lecce 73100, Italy. E-mail: riccardo.dicorato@cnr.it
hCenter for Biomolecular Nanotechnologies, Istituto Italiano di Tecnologia, Arnesano 73010, Italy
First published on 8th March 2023
The application of superparamagnetic iron oxide nanoparticles (SPIONs) in drug delivery, magnetic resonance imaging, cell tracking, and hyperthermia has been long exploited regarding their inducible magnetic properties. Nevertheless, SPIONs remain rapidly cleared from the circulation by the reticuloendothelial system (RES) or mononuclear phagocyte system, with uptake dependent on several factors such as the hydrodynamic diameter, electrical charge and surface coating. This rapid clearance of SPION-based theranostic agents from circulation is one of the main challenges hampering the medical applications that differ from RES targeting. This work proposes a strategy to render biocompatible SPIONs through their encapsulation in the red blood cells (RBCs). In this work, the research has been focused on the multi-step optimization of chemical synthesis of magnetic nanoparticles (MNPs), precisely iron oxide nanoparticles (IONPs) and zinc manganese-ferrite nanoparticles (Zn/Mn FNPs), for encapsulation in human and murine RBCs. The encapsulation through the transient opening of RBC membrane pores requires extensive efforts to deliver high-quality nanoparticles in terms of chemical properties, morphology, stability and biocompatibility. After reaching this goal, in vitro experiments were performed with selected nanomaterials to investigate the potential of engineered MNP-RBC constructs in theranostic approaches.
In this way, the chemical formulation of nanoparticles with a compound without a regulatory status (i.e. excipients, organic solvents, new synthetic polymers, etc.) makes the development process more challenging, longer and, therefore, more expensive.9 The complexity and reproducibility of the preparative method of nanoparticles are also important aspects that must be considered since they could hinder the continuity of the project towards clinical phases.
The current development in medical imaging technology aims for early diagnosis and consequently, timely and personalized treatment of the pathology, which often implies the visualization of specific targets in the diseased organism by labeling with tracer agents such as magnetic iron oxide nanoparticles that have been extensively explored especially as magnetic resonance imaging (MRI) tracers through control over their size, composition and morphology.10 Although advances in nanotechnology have improved the stability and biocompatibility of iron oxide-based contrast agents, the phagocytic system still limits the applicability of these tracers that have a blood half-life range of 1–3 h and the time window for bolus-based measurements is only a few minutes.11 Moreover, several iron oxide contrast agents were discontinued due to safety concerns.12
In the last few years, a strategy has been developed based on the encapsulation of some SPIONs, mainly commercial, and/or USPION contrast agents in red blood cells (RBCs) as a useful alternative to overcome the limitations described above.13 When placed in a hypotonic solution, human erythrocytes have a remarkable capacity to undergo reversible membrane swelling with the simultaneous appearance of pores large enough to be crossed by metabolites and macromolecules; this property has been carefully investigated for a long time in laboratories, and resealed erythrocytes have been proposed as carriers and bio-reactors suitable for use in the treatment of various diseases.14,15 In fact, RBCs have potential to act as biocompatible carriers for several compounds16–20 and contrast agents since they survive in circulation for weeks, therefore permitting a potential extension of the time window for imaging in diagnostic applications. In the last few years, some efforts have been made to identify, among commercially available and preclinical SPIONs, those that are efficiently encapsulated in RBCs.21 Indeed, not all iron oxide nanoparticles can be efficiently loaded into RBCs and their encapsulation varies according to several factors, namely the synthesis protocol, the dispersant agent and the nanoparticle size. Moreover, if SPIONs are not stabilized properly, the particle suspensions may aggregate or the particle core and coating may degrade. Hence, to be able to use RBCs as an effective diagnostic tool in this field, there is an urgent need to identify magnetic nanomaterials that are potentially suitable for producing SPION-loaded RBCs with long half-lives in blood circulation. Such SPION-loaded RBCs could open up a variety of medical applications that require intravascular magnetic tracers.
In this work we focused our attention on the possibility to encapsulate newly synthesized magnetic nanoparticles (MNPs) with different chemico-physical characteristics, into human RBCs through the open membrane pores without affecting cell viability. The main challenge in producing nanoparticles for the engineering of RBCs is represented by the pathway of the material internalization: unlike other mammalian cells (that take up many kinds of nanomaterials with different shapes and sizes by a well-defined mechanism of endocytosis or phagocytosis22,23), the nanoparticle loading into RBCs is only related to the passage through transient opened pores on the cell membrane. Consequently, the morphology, monodispersity and stability of the nanomaterial become critical aspects for the encapsulation of SPIONs in RBCs. The immediate perspective of this work is optimization of the stability of the nanoparticle suspensions for enhancing the encapsulation rate into the RBCs with the future perspective of characterizing their magnetic behavior in related applications. For example, SPION-RBC constructs could represent new intravascular contrast agents for some diagnostic applications, such as MRI/fMRI and magnetic particle imaging (MPI).24
In order to obtain high quality SPIONs, there are several specific parameters to set, which must be strictly followed in order to tailor the magnetic properties to the aimed application and gain maximum efficiency and no side effects in humans.25,26 Herein, the preparation of different MNPs based on iron oxide or zinc manganese-ferrite nanoparticles is described. Synthetic strategies such as the thermal decomposition route are still not ideal to achieve highly loaded RBCs since these nanoparticles need an additional processing to be transferred in water,27 with a low-yield in terms of the final concentration. Therefore, the strategy here was to optimize the well-established co-precipitation method, and reach a high yield of concentrated nanoparticles and further RBC loading.28,29 Temperature, nucleation and growth time, filtration, and coating were finely adjusted to deliver the most suitable nanomaterials for encapsulation in the RBCs. The biological parameters of the selected best engineered MNP-RBC constructs as well as the efficiency of magnetic nanoparticle encapsulation and their potential as magnetic carriers eligible for theranostic applications were evaluated.
For the preparation of the second series of samples, both the salt solution and ammonium hydroxide were maintained at the same temperatures (MNPs 5–7). This approach leads to the non-controlled formation of some clusters during the reaction, as can be seen from DLS measurements, which implies a slight polydispersity. These nanoparticles were much larger, less uniform and more aggregated than typical spherical nanoparticles, with a similar size trend compared to the first batch. Finally, with the third series of nanoparticles (MNPs 8–10) the ammonium hydroxide was kept at room temperature whereas the temperature of the saline solution was tuned. The effect of the temperature was confirmed, observing a reduction of particle size correlated with the increase of the reaction temperature. The crystal size is reduced when the temperature is blocked at 40 °C (MNPs 9); nanoparticles show a good hydrodynamic size distribution (25–35 nm by DLS analysis) when the salt solution was heated to 40 °C and the ammonium hydroxide (NH4OH) was kept at 25 °C. TEM measurements showed an average core diameter between 7 nm and 12 nm (Fig. 1).
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Fig. 1 TEM images of sample MNPs 3 (a), MNPs 6 (b) and MNPs 9 (c), obtained by varying the temperature of the precursor solutions. All scale bars correspond to 20 nm. |
Samples | [Dextran] (M) | DLS size (nm) | PDI |
---|---|---|---|
MNPs 14 | 3.7 | 125.2 | 0.184 |
MNPs 15 | 7.4 | 107.8 | 0.168 |
MNPs 16 | 3 | 61.45 | 0.156 |
MNPs 17 | 1.5 | 67.84 | 0.223 |
MNPs 18 | 0.75 | 74.07 | 0.139 |
MNPs 19 | 0.375 | 65.06 | 0.213 |
MNPs 20 | 0.187 | 34.04 | 0.214 |
MNPs 21 | 0.093 | 58.05 | 0.392 |
MNPs 22 | 0.047 | 104.9 | 0.316 |
The dextran molarity has been calculated with the final reaction volume of 15 mL and considering the molecular weight of the glucose monomer (180.16 Da).
FT-IR measurements were performed on prepared nanoparticles to assess the presence of the different coatings (Fig. 3c). The ATR spectra of sodium citrate and dextran were acquired directly by analyzing the commercial powders. The characteristic bands of COO− at 1583 cm−1 (antisymmetric stretching) and 1417 cm−1 (symmetric stretching) were detected for sodium citrate; both bands were revealed in the citrate-coated MNPs, with a red shift of the symmetric stretching to 1396 cm−1. Concerning the dextran, two main functional groups were detected: the stretching vibration of C–O–C (at 1149, 1105 and 1003 cm−1) and the vibration modes related to υ(C–H) (at 1415 and 1344 cm−1); the same characteristic peaks were revealed in the dextran-coated MNPs. In this sample, the peaks related to citrate molecules were not detected at all, confirming the ligand exchange procedure at the surface of the nanoparticles. In all the MNP-spectra a peak at 555–560 cm−1 was observed, related to the Fe–O bond. Moreover, in all the spectra (except sodium citrate) a large shoulder between 3100 and 3500 cm−1 was observed for the stretching of OH. Finally, for nude iron oxide and for both the samples containing dextran, an intense peak in the range 1600–1650 was detected, attributed to the vibration of H–O–H (δO–H) of coordinated water molecules.
Samples | Crystal growth | [Fe] (mM) | DLS size (nm) | PDI |
---|---|---|---|---|
MNPs 28 | 30 s | 996.9 | 40.34 | 0.127 |
MNPs 29 | 60 s | 881.0 | 34.43 | 0.158 |
MNPs 30 | 120 s | 652 | 27.61 | 0.274 |
MNPs 31 | 300 s | 1887 | 43.98 | 0.268 |
For a comparison between the different suspensions of nanoparticles, the sample MNPs 20 was synthesized by using the classic method where no citric acid was used to stop the 3 h-growth step.
The obtained nanoparticles had a smaller and more regular shape than MNPs 20, as measured by TEM analysis observation (Fig. 4). Moreover, the samples obtained at 30 and 60′′ showed a narrow hydrodynamic diameter and a very low polydispersity, by DLS analysis. The colloidal stability of such a formulation was excellent, with the absence of any sedimentation for months at room temperature.
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Fig. 4 TEM images of iron oxide MNPs obtained with crystal growth steps of 30′′ (MNPs 28, panel a), 60′′ (MNPs 29, panel b) and 120′′ (MNPs 30, panel c). All scale bars correspond to 50 nm. |
First, to establish the most suitable amount of elements used for the synthesis, different molar ratios between FeCl2, FeCl3, MnCl2 and ZnCl2 were applied. Hence, the amount of FeCl3 (2.8 mmol) was maintained constant, while that of FeCl2 (1.6 mmol) was reduced to 0.8 mmol. This means that the other 0.8 mmol of XCl2 salt (where X is Zn or Mn) was replaced with different amounts of zinc and/or manganese (Table 3). The chemical analysis was performed using inductively coupled plasma (ICP), which confirmed the elemental composition used for all the samples synthesized.
Samples | MnCl2 (mmol) | ZnCl2 (mmol) | FeCl2 (mmol) | FeCl3 (mmol) | DLS size (nm) | PDI |
---|---|---|---|---|---|---|
All the reported analyses referred to citrate-coated nanoparticles. | ||||||
MNPs 32 | 0 | 0 | 1.6 | 2.8 | 45.21 | 0.168 |
MNPs 33 | 0 | 0.8 | 0.8 | 2.8 | 36.86 | 0.227 |
MNPs 34 | 0.16 | 0.64 | 0.8 | 2.8 | 92.24 | 0.401 |
MNPs 35 | 0.32 | 0.48 | 0.8 | 2.8 | 43.19 | 0.190 |
MNPs 36 | 0.48 | 0.32 | 0.8 | 2.8 | 25.57 | 0.196 |
MNPs 37 | 0.64 | 0.16 | 0.8 | 2.8 | 38.93 | 0.346 |
MNPs 38 | 0.8 | 0 | 0.8 | 2.8 | 59.25 | 0.198 |
After an accurate study of the morphology of nanoparticle suspensions, an evident difference was observed between the sample MNPs 36 and the other remaining samples. TEM analysis (Fig. S2†) showed that the sample MNPs 36 presented a more defined and quasi-spherical shape. Regarding the other samples, they were much larger and less uniform than the sample MNPs 36, with some nanoparticles reaching a hydrodynamic diameter of 80–90 nm by DLS, with a higher polydispersity index.
The magnetic properties of the samples MNPs 32–MNPs 38 were systematically investigated by recording the temperature dependent magnetization curves at 298 K and 5 K (Fig. 5). The samples at 298 K are all in the superparamagnetic regime, showing coercive fields (Hc) smaller than 5.1 kA m−1 (Fig. 5a and Table 4). The highest saturation magnetization (Ms) values at 298 K were achieved by the MNPs 32 sample (pure iron oxide) with a value of 107.5 Am2 kg−1, followed by the samples doped with both Mn and Zn with Ms values between 66 and 73 Am2 kg−1. The samples doped with only Zn or Mn showed the lowest values of Ms (40.3 e 55.8 Am2 kg−1, respectively). The magnetization measurements at 5 K showed larger values matching the trend observed at 298 K with also larger coercivity field (Hc), confirming the suitability of these nanoparticles for biological applications.
Sample | T = 298 K | T = 5 K | ||||
---|---|---|---|---|---|---|
M s (Am2 kg−1) | M r (Am2 kg−1) | H c (kA m−1) | M s (Am2 kg−1) | M r (Am2 kg−1) | H c (kA m−1) | |
MNPs 32 | 107.5 | 6.0 | 5.1 | 127.7 | 33.1 | 20.1 |
MNPs 33 | 40.3 | 0.3 | 1.6 | 56.6 | 12.6 | 35.1 |
MNPs 34 | 73.0 | 2.6 | 2.6 | 96.3 | 19.8 | 23.5 |
MNPs 35 | 71.4 | 2.7 | 2.7 | 90.7 | 23.3 | 15.5 |
MNPs 36 | 67.9 | 2.0 | 2.0 | 98.9 | 23.1 | 16.0 |
MNPs 37 | 66.1 | 2.1 | 2.1 | 90.3 | 26.4 | 18.1 |
MNPs 38 | 55.8 | 0.9 | 1.5 | 79.4 | 23.2 | 27.0 |
Considering the results achieved by tuning the growth phase for iron oxide nanoparticles, a similar approach has been tested for Zn/Mn FNPs, by stopping the growth of the crystal core by adding a citric acid solution. Interestingly, a growth time of 60′′ (ideal condition for iron oxide) was not sufficient to obtain a stable nanoparticle suspension. Therefore, the reaction time for the ferrites was increased to 90′′ (MNPs 39), 120′′ (MNPs 40) and 300′′ (MNPs 41), as reported in Table 5 and in Fig. 6. The sample MNPs 36DX was obtained by applying a standard 3 hours step of reaction and it was used for comparison with the fast-reaction samples.
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Fig. 6 TEM images of Zn/Mn FNPs obtained with crystal growth steps of 90′′ (MNPs 39, panel a), 120′′ (MNPs 40, panel b) and 300′′ (MNPs 41, panel c). All scale bars correspond to 50 nm. |
Sample | Crystal growth | [Fe] (mM) | DLS size (nm) | PDI |
---|---|---|---|---|
All the reported analyses referred to dextran-coated nanoparticles. | ||||
MNPs 39 | 90 s | 469.6 | 32.16 | 0.292 |
MNPs 40 | 120 s | 288.3 | 43.02 | 0.114 |
MNPs 41 | 300 s | 319 | 44.75 | 0.355 |
MNPs 36DX | 3 hours | 1449 | 48.59 | 0.144 |
These nanoparticles presented a very high colloidal stability even after 1 year of storage at room temperature. As shown in Fig. S3,† the DLS analysis of MNPs 29 and MNPs 40 resulted in similar profiles, with negligible variations (around 10%) in size and PDI.
To evaluate the heating potential of the synthesized nanoparticles, two approaches were explored: photothermia and magnetic hyperthermia. For both techniques, the heat-generating capacity of the nanoparticles (specific absorption rate, W gFe−1) was normalized by the MNP iron concentration. For photothermia, the heating performances decreased with increasing concentration of nanoparticles in the suspension, as previously reported.33 Both formulations based on iron oxide (MNPs 20 and 29) show higher SAR values than the Ferucarbotran® used as a standard. Even higher values were obtained with the MNPs 36 and 40 Zn/Mn FNPs (Fig. 7d). Interestingly, both samples obtained with the fast growth method, MNPs 29 and 40, proved to be more efficient than the equivalents obtained with the traditional growth technique (MNPs 20 and 36), confirming the effectiveness of the method used. Regarding magnetic hyperthermia (Fig. 7e), the iron oxide nanoparticles showed performances comparable with the Ferubarbotran® standard. In contrast, the Zn/Mn FNPs did not produce satisfactory results in terms of SAR and therefore were not suitable for the application of this protocol.
The first chemical syntheses have led to different new nanoparticle suspensions (MNPs) composed of iron oxides and/or ferrites containing manganese and zinc with different physicochemical characteristics. All nanoparticle suspensions were tested for encapsulation in the human red blood cells (RBCs) using a hypotonic dialysis method.21 An automated hemocytometer has been used to assess biological parameters of MNP-loaded RBCs obtained at the end of the loading procedure, in comparison with control cells, Table 6. The final recovery of these MNP-RBC constructs, expressed as the percentage of cells obtained with respect to those utilized during the dialysis step, was also calculated. Table 6 shows also the values of longitudinal (T1) and transverse (T2) relaxation times obtained by NMR measurements of all MNP-RBC-loaded samples.
Samples | Metal ions | Coating | T1 (ms) | T2 (ms) | MCV (fl) | MCH (pg) | MCHC (g dl−1) | Cell recovery (%) |
---|---|---|---|---|---|---|---|---|
MNP–RBC loaded samples were prepared by using 11.2 mg Fe of each colloidal suspension added to 1 mL of RBCs at 70% of hematocrit during the dialysis step of the loading procedure. Values were compared to those of control cells (ND-RBCs; not dialysed RBCs and UL-RBCs; unloaded-RBCs). | ||||||||
ND-RBCs | — | — | 2210 | 63 | 88 | 31 | 34.3 | — |
UL-RBCs | — | — | 2080 | 63 | 76 | 22.2 | 29.1 | 70 |
MNPs 11CIT-filt.0.22-RBCs | Fe | Citrate | 40.9 | <5 | 67 | 51.9 | 77.9 | 48 |
MNPs 14DX-non filt.-RBCs | Fe | Dextran | 490 | 4.9 | 82 | 26.4 | 32.2 | 49 |
MNPs 20DX-non filt.-RBCs | Fe | Dextran | 93.4 | 12.6 | 69 | 25.3 | 36.4 | 48 |
MNPs 20CHI-non filt.-RBCs | Fe | Chitosan | — | — | 84 | 9.9 | 11.9 | 0 |
MNPs 35CIT-filt.0.22-RBCs | Fe/Mn/Zn | Citrate | — | — | 76 | 10.7 | 14 | 21 |
MNPs 36CIT-filt.0.22-RBCs | Fe/Mn/Zn | Citrate | 23.7 | 10.7 | 63 | 19.9 | 31.3 | 39 |
Despite the data shown in Table 6 demonstrating a significant decrease in T1 and T2 values of RBCs-MNPs compared to the values of control samples (ND, not dialyzed RBCs; 2210 ms, and UL, unloaded RBCs, 2080 ms), TEM analyses have shown that these nanoparticle formulations are not suitable for encapsulation in the human red blood cells (Fig. 8).
In fact, representative samples such as MNPs 36CIT-filt 0.22 RBCs (Fig. 8a), MNPs 20DX-non filt-RBCs (Fig. 8b), and MNPs 20CHIT no filt-RBCs (Fig. 8c) reveal the presence of abundant iron oxides only in the extracellular spaces. Therefore, despite a strong decrease of T1 and T2 values being evidenced (Table 6) which would indicate the presence of iron oxide amounts (even higher for some samples) in the human RBC samples, the nanomaterials were not really encapsulated.
Moreover, it appears that chitosan is the least suitable coating agent to permit the encapsulation of nanoparticles in the RBCs.
In fact, the use of MNPs 20CHI-non filt leads to complete cell lysis (and this also when low iron amounts such as 2.8 mg mL−1 RBCs 70% are used, data not shown) evidencing that not all surfactant agents are eligible to preserve the biocompatibility of the nanoparticle formulation (Table 6 and Fig. 8). Furthermore, the final cell recovery of MNP-loaded RBC samples was significantly lower (ranging from 0 to 49%) compared to the control unloaded RBC sample (UL-RBCs, 70%). The data obtained with the first nanoparticle formulations allowed us to understand how to modify the chemical synthesis protocol, which also depends on the ability of nanomaterials to be more efficiently encapsulated in the RBCs.
In a second step, other nanoparticle suspensions were synthesized according to the nucleation time and multiple-filtrations (as reported in sections 3.4 and 3.5) in order to improve their ability to be loaded in the erythrocytes. Table 7 shows some biological parameters (such as MCV, MCH, and MCHC) and T1 and T2 values obtained by NMR measurements of these MNP-RBC-loaded samples.
Samples | T1 (ms) | T2 (ms) | MCV (fl) | MCH (pg) | MCHC (g dl−1) | Cell recovery (%) |
---|---|---|---|---|---|---|
Samples were characterized by T1 and T2 NMR measurements and evaluation of some biological properties (ND-RBCs; not dialysed RBCs and UL-RBCs; unloaded-RBCs; MNP-loaded RBCs were prepared using 11.2 mg Fe per mL of RBCs 70% Ht). | ||||||
ND-RBCs | 2236 | 57.7 | 88 | 29.3 | 33.4 | — |
UL-RBCs | 2192 | 54.5 | 75 | 22.6 | 31.3 | 70 |
MNPs 26DX-non filt.-RBCs | 836.5 | 5.3 | 52 | 13.4 | 25.6 | 67 |
MNPs 25DX-filt.0.22-RBCs | 452.4 | <5 | 56 | 13.6 | 24.2 | 53 |
MNPs 20DX-filt.0.22-RBCs | 541.7 | <5 | 68 | 20 | 29.3 | 50 |
MNPs 36DX-filt.0.22-RBCs | 414.6 | 8.3 | 75 | 19.6 | 26.1 | 43 |
MNPs 35DX-non filt.-RBCs | 556.4 | 10.3 | 74 | 15.1 | 20.4 | 44 |
MNPs 35DX-filt.0.22-RBCs | 503.8 | 8.8 | 81 | 22.8 | 28.2 | 48 |
MNPs 20 DX-filt.0.1 -RBCs | 664.4 | <5 | 67 | 18.7 | 27.8 | 61 |
MNPs 36 DX-filt.0.1 -RBCs | 652 | 11.7 | 83 | 21.9 | 26.3 | 60 |
The MNP-loaded RBC samples present a cell volume similar to control cells (ND- and UL-RBCs) except for MNPs 26DX-non filt.-RBC and MNPs 25DX-filt.0.22-RBC samples that are much smaller (MCV; 52 and 56 fl, respectively). Moreover, these two last samples have less mean hemoglobin concentration (MCH) than the other MNP-loaded samples. All samples have lower cell recovery (ranging from 43 to 67%) with respect to the control sample (UL-RBCs; 70%) at the end of the procedure.
TEM analyses (Fig. 9) permitted us to establish which nanomaterials are more suitable for their encapsulation in the erythrocytes as not in all cases a strong decrease of T1 and T2 values corresponded to the effective entrapment of iron oxide into cells.
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Fig. 9 TEM images of slices of human RBCs loaded with (a) MNPs 35DX-filt.0.22 and (b) MNPs 36DX-filt.0.1. Additional TEM images concerning the effect of filtration on sample MNPs 20 are reported in Fig. S4.† |
Particularly, MNP35DX-filt.0.22-RBCs (Fig. 9a) and MNP36DX-filt.0.1-RBCs (Fig. 9b) showed the presence of iron oxides; however, MNP36DX-filt.0.1-RBCs evidenced the iron oxide nanomaterial in monodisperse form and only into cells, while in the MNP35DX-filt.0.22-RBC sample, iron oxide clusters were found both in the cells and in the extracellular space.
It appears that magnetic nanoparticles processed with 0.1 μm filters, such as MNPs 20DX-filt.0.1 and MNPs 36DX-filt.0.1, have enabled achieving cell recoveries (60–65%) similar to those of control cells (UL-RBCs) at the end of the loading procedure (Table 7). However, the results showed that the MNPs 36DX-filt.0.1 nanoparticle suspension appears the most eligible among the nanoparticle formulations using the 0.1 μm filter to maintain the native properties of RBCs at the end of the loading procedure.
Concerning the formulations processed with the 0.22 μm filter, a higher decrease of T1 and T2 values was observed (for example for MNPs 25DX-filt.0.22 and MNPs 36DX-filt.0.22 samples, Table 7), but lower final cell recovery of loaded RBCs was found (43–53%).
Afterwards, the nanoparticles obtained with a very short growth phase (see section 3.6 above) were tested for the loading procedure with human RBCs and the results are reported in Table 8. It appears that a growth phase of 0.5–5 minutes during the synthesis process of nanoparticles, in combination with 0.1 μm filtration, and providing formulations such as MNPs 41 lead to nanoparticle formulations (both iron oxide-MNPs and ferrite-MNPs) with greater ability to be encapsulated in the RBCs (Fig. 10). However, not all these nanoparticle formulations are very efficiently encapsulated (Fig. 10), despite a remarkable decrease of T1 and T2 values (e.g. for MNPs 28DX-filt 0.1, MNPs 30DX-filt 0.1 and MNPs 39DX-filt 0.1-loaded RBC samples) being evidenced.
Samples | T1 (ms) | T2 (ms) | Fe [mM] | MCV (fl) | MCH (pg) | MCHC (g dl−1) | Cell recovery (%) |
---|---|---|---|---|---|---|---|
The samples were prepared by using 11.2 mg Fe per mL RBCs at 70% of hematocrit. Results were compared to those of unloaded RBCs (UL-RBCs) obtained by the same procedure with the exception that they were dialysed in the absence of the magnetic material. | |||||||
UL-RBCs | 2135 | 67.5 | — | 75 | 22 | 29.2 | 70 |
MNPs 28DX-filt 0.1-RBCs | 150 | 6.7 | 4.75 | 72 | 44.3 | 61.3 | 56 |
MNPs 29 DX-filt 0.1 -RBCs | 394.3 | <5 | 1.87 | 70 | 19.5 | 27.9 | 60 |
MNPs 30DX-filt 0.1-RBCs | 532.2 | <5 | 1.1 | 78 | 19.8 | 25.4 | 51 |
MNPs 31DX-filt 0.1-RBCs | 726 | 5.2 | 0.71 | 82 | 22.5 | 27.5 | 49 |
MNPs 39DX-filt 0.1-RBCs | 351.6 | 5.4 | 1.82 | 68 | 12.3 | 18.2 | 59 |
MNPs 40 DX-filt 0.1 -RBCs | 141.9 | <5 | 2.66 | 75 | 19 | 25.6 | 47 |
MNPs 41DX-filt 0.1-RBCs | 297.9 | <5 | 2.2 | 76 | 16.4 | 21.5 | 52 |
Therefore, the chemical synthesis of nanoparticles with a very reduced growth phase (order of minutes) appears to lead to an improvement of encapsulation into cells, of both pure iron oxide formulations, such as MNPs 30DX-filt 0.1, and specific ferrite formulations such as MNPs 41 nanoparticle suspension, although, in some cases, the MNP-loaded RBC samples still present some iron clusters outside of cells (Fig. 10d).
TEM analyses have permitted us to continue the experiments with selected best type of nanoparticle, namely iron oxide-MNPs 29DX-filt 0.1 and ferrite-MNPs 40DX-filt 0.1. These specific nanoparticles are efficiently encapsulable in the human RBCs; both magnetic nanoparticle suspensions are present inside the cells and also evenly distributed in the cytoplasm and, moreover, in the MNPs 29- and MNPs 40-loaded RBC samples there are no iron oxides adhering to the cell surface or in the extracellular spaces (Fig. 11c and d for MNPs 29DX-filt 0.1 and e and f for MNPs 40DX-filt 0.1).
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Fig. 11 TEM images of RBCs (whole cells in a, c, and e; slices in b, d, and f) obtained at the end of the loading procedure by using MNPs 29DX-filt 0.1 (c and d) and MNPs 40DX-filt 0.1 (e and f) nanoparticles. Unloaded-RBCs are reported in panels (a) and (b). TEM images of non-dialyzed RBCs (ND-RBCs) are shown in Fig. S6.† |
Moreover, MNPs 29 and MNPs 40-loaded RBC samples maintain the native cell morphology when compared to control samples (UL-RBCs, Fig. 11a and b). Electron microscopy analyses validate the iron concentration (mM) obtained from NMR measurements and by using calibration curves for MNPs 29 and MNPs 40 nanoparticles, respectively. The remarkable decrease of T1 and T2 values (e.g. T1 for MNPs 29-loaded RBCs, 394.3 ms; T1 for MNPs 40-loaded RBCs, 141.9 ms, Table 8) with respect to the control value (UL-RBCs, 2135 ms) corresponds to effective entrapment of iron oxides in the cells. The calibration curves for MNPs 29 and MNPs 40 nanoparticles were recorded using RBC suspensions at 44% of hematocrit. The values of 1/T1c–1/T10 and of 1/T2c–1/T20 have been used to obtain the values of longitudinal and transverse relaxivities (r1 = 1.1379 and r2 = 180.02, respectively, for MNPs 29 and r1 = 2.5176 and r2 = 97.643, respectively, for MNPs 40 (Fig. 12)). Essentially, calibration curves were obtained as reported in the ESI for the Ferucarbotran® contrast agent (Fig. S5†). By using r1 values, the amounts of iron present in the MNPs 29- and MNPs 40-loaded RBC samples were calculated; mM Fe concentrations, obtained by using a range of 2.8–22.4 mg Fe per mL RBCs 70% during the loading procedure, are reported in Table 9. The data show that the efficiency of iron encapsulation for both nanomaterials increases with the increased iron amounts used; an encapsulation of iron ranging from 0.6 ± 0.1 to 1.9 ± 0.4 mM Fe for MNPs 29-loaded RBCs and from 0.5 ± 0.1 to 2.8 ± 0.1 mM Fe for and MNPs 40-RBCs was obtained. Moreover, the data have indicated that the cell morphology and biological properties of the MNPs 29- and MNPs 40-loaded RBCs are not significantly different from those of native cells (Table 9). Only at higher iron amounts used for the loading procedure a slight decrease of MCH and MCHC values occurs. These preliminary experiments have evidenced the suitability of iron oxide-MNPs 29 and ferrite-MNPs 40 nanoparticles for encapsulation in human RBCs despite the final RBC-constructs showing different cell recovery values at the end of the loading procedure (MNPs 40-loaded RBCs lower than MNPs 29-loaded RBCs, Table 9).
Samples | T1 (ms) | T2 (ms) | Fe [mM] | MCV (fl) | MCH (pg) | MCHC (g dl−1) | Cell recovery (%) |
---|---|---|---|---|---|---|---|
UL-RBCs | 2069 ± 15.6 | 62.1 ± 1.3 | — | 77.5 ± 0.7 | 25.2 ± 0.6 | 34.3 ± 1.3 | 70.1 ± 0.1 |
L1-MNPs 29DX-filt 0.1 | 939 ± 270 | 6.9 ± 0.9 | 0.6 ± 0.1 | 74 ± 4.2 | 25.4 ± 0.3 | 35.2 ± 1.5 | 62 ± 2.8 |
L2-MNPs 29DX-filt 0.1 | 733 ± 173 | 6 ± 0.5 | 1 ± 0.3 | 74 ± 0.7 | 25 ± 0.6 | 34 ± 0.8 | 57 ± 12 |
L3-MNPs 29DX-filt 0.1 | 481.5 ± 123 | <5 | 1.5 ± 0.5 | 74 ± 5.7 | 21.8 ± 3.2 | 29.4 ± 2.1 | 54.9 ± 7.3 |
L4-MNPs 29DX-filt 0.1 | 402.5 ± 70.2 | <5 | 1.9 ± 0.4 | 70.5 ± 6.4 | 20.3 ± 3.6 | 28.6 ± 2.5 | 54.3 ± 10.9 |
L1-MNPs 40DX-filt 0.1 | 590 ± 64.1 | 9.3 ± 0.4 | 0.5 ± 0.1 | 76.5 ± 3.5 | 25.1 ± 0.3 | 33 ± 1.9 | 53.6 ± 5.1 |
L2-MNPs 40DX-filt 0.1 | 373.3 ± 42.5 | 5.7 ± 0.4 | 0.9 ± 0.1 | 75.5 ± 0.7 | 24.7 ± 0.8 | 32.8 ± 0.8 | 52.2 ± 5.9 |
L3-MNPs 40DX-filt 0.1 | 196.5 ± 77.2 | <5 | 2 ± 0.9 | 74.5 ± 0.7 | 21.4 ± 3.3 | 28.6 ± 4.2 | 46.5 ± 0.7 |
L4-MNPs 40DX-filt 0.1 | 135 ± 5.2 | <5 | 2.8 ± 0.1 | 74.5 ± 2.1 | 18.9 ± 1.1 | 25.3 ± 2.5 | 42 ± 1.4 |
On the other hand, the cell recovery of MNPs 29- and MNPs 40-loaded human RBCs obtained at the end of the procedure decreases when higher amounts (L4) of nanoparticles are used (from 62 ± 2.8% to 54.3 ± 10.9% for MNPs 29-RBCs and from 53.6 ± 5.1% to 42 ± 1.4% for MNPs 40-RBC samples, Table 9). TEM images showed iron oxides inside the cells evenly distributed in the cytoplasm of MNPs 29- and MNPs 40-loaded RBCs. However, in some cases, the presence of a few iron oxide clusters was also found in cells and particularly in MNPs 29-loaded RBCs (Fig. 11).The data obtained so far indicate that the ferrite MNPs 40 nanoparticles have a higher encapsulation efficiency (L4-MNPs 40-loaded RBC; 2.8 ± 0.1 mM Fe) than the iron oxide MNPs 29 nanoparticles (L4-MNPs 29-loaded RBCs 1.9 ± 0.4 mM Fe). However, it should be considered that the final cell recovery remarkably decreases. We also tested these nanomaterials with murine red blood cells in order to understand if an in vivo application of these MNP-loaded RBC constructs can be considered. Table S4† and Table 10 report representative MNPs 29- and MNPs 40-loaded RBCs obtained by using human and murine red blood cells and 11.2 mg Fe per mL RBCs 70% (to obtain L3-loaded samples), respectively. The data obtained at the end of the loading procedure have indicated that typical biological properties of murine RBCs were maintained (Table 10). The cell recovery of murine RBCs at the end of the loading procedure appears similar for MNPs 29- and MNPs 40-RBC constructs (34.3 ± 8.1% and 32.3 ± 6.7%, respectively) and both values are slightly lower than the values obtained for the control sample (44.2 ± 5.4% for the UL-RBCs).
Samples | T1 (ms) | T2 (ms) | Fe [mM] | MCV (fl) | MCH (pg) | MCHC (g dl−1) | Cell recovery (%) |
---|---|---|---|---|---|---|---|
Samples obtained by using 11.2 mg Fe per mL RBC70%. Values are expressed as means ± SD of three similar experiments. | |||||||
UL-RBCs | 2078.5 ± 108 | 57.4 ± 4.8 | — | 41.5 ± 0.7 | 15.9 ± 0.6 | 38.5 ± 1.9 | 44.2 ± 5.4 |
L3-MNPs 29 | 484.4 ± 26.8 | 5.9 ± 2.1 | 1.90 ± 0.02 | 41.5 ± 0.7 | 14.9 ± 0.6 | 35.6 ± 0.9 | 34.3 ± 8.1 |
L3-MNPs 40 | 440.9 ± 92.3 | 7.8 ± 2.7 | 0.90 ± 0.20 | 41.0 ± 2.8 | 13.8 ± 1.3 | 33.8 ± 0.9 | 32.3 ± 6.7 |
L3-Ferucarbotran | 177 ± 27.60 | <5 | 4.05 ± 0.83 | 40 ± 0.7 | 11.75 ± 1.06 | 31.5 ± 0.8 | 43 ± 8.3 |
As expected, murine RBCs are smaller in diameter, size and volume and permit encapsulation of lower MNP amounts into human RBCs; on the other hand, they are more fragile and the cell recovery at the end of the procedure is lower with respect to that obtained by using human RBCs.
However, the in vitro results with murine RBCs reported in Table 10 are essential to evaluate the possibility to perform in vivo experiments. The murine model permits us to study the survival in the blood circulation of these new MNP-RBC constructs in forthcoming in vivo studies on best-performing MNP-RBC constructs.
The decrease of T1 and T2 values, obtained by the NMR measurements, indicated a possible encapsulation of these nanomaterials also in murine RBCs. By using the calibration curves (Fig. 13), specifically obtained for MNPs 29 and MNPs 40 nanoparticles, it was possible to calculate mM Fe concentrations present in MNPs 29- and MNPs 40-loaded samples (Table 10). The resulting mM iron concentrations entrapped in murine MNPs 29- and MNPs 40-loaded RBCs, calculated using r1 values derived from calibration curves, were 1.90 ± 0.02 mM and 0.90 ± 0.20 mM, respectively, for MNPs 29- and MNPs 40-RBC constructs. It is evident that the encapsulation of the Ferucarbotran® contrast agent (gold standard) was more efficient when both human and murine RBCs were used (Table 10 and Table S4†).
Anyway, these preliminary studies have indicated that the iron oxide nanoparticles such as MNPs 29 and ferrite-MNPs 40 nanoparticles, are also encapsulable in the murine RBCs (Fig. 14). These data are promising to continue the validation of these nanomaterials for the loading in the murine erythrocytes with the aim to perform future in vivo experiments through the use of a murine animal model.
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Fig. 14 TEM images of whole-murine RBCs obtained at the end of the loading procedure by using Ferucarbotran (a and b), MNPs 29DX-filt 0.1 (c and d) and MNPs 40DX-filt 0.1 (e and f) nanoparticles. Additional TEM images of Ferucarbotran-loaded human RBCs are reported in Fig. S7.† |
The MPS signal was acquired upon application of a sinusoidal magnetic field as reported in the Experimental section. The MPS results of free MNPs and MNP-loaded RBCs were verified against the measurement of unloaded RBCs as well as an empty signal measurements, which mimic the noise level of the MPS. Thus, the results are valid up to the frequency at which their responses cross the response of the empty reference signal. In order to investigate the difference between the RBC signal and the signal of the immobilized particles, the spectral responses are normalized to the immobilized signal. The results for the MNPs 29- and the MNPs 40 particles are shown in Fig. 15. MNPs 29-loaded RBCs give a frequency signal which is slightly weaker than the signal of their immobilized counterparts, by approximately half an order of magnitude. The two signals run in a parallel manner along the higher harmonics, up to a frequency of 750 kHz, at which the signals start to vanish in the noise. This shows that the RBC signal is very close to the signal of immobilized MNPs 29 and differs only by a frequency constant scaling factor (Fig. 15a). For the MNPs 40-loaded RBCs the measurements show a similar result (Fig. 15b).
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Fig. 15 MPS spectra of representative human L3-MNPs 29DX-filt 0.1-RBCs (a) and L3-MNPs 40DX-filt 0.1-RBCs (b) samples in comparison with the respective immobilized and liquid nanoparticles. |
Moreover, the MPS signal runs parallel to the signal of the immobilized MNPs 40 particles over the first higher harmonics. As of 400 kHz, the signal starts to fluctuate more compared to the signal of the immobilized particles. Interestingly, the free particle signal shows the same fluctuation. Again, the measurements start to vanish in the noise at approximately 750 kHz. Therefore, it appears that MNPs 29-RBC constructs perform slightly better than MNPs 40-RBCs when evaluated with MPS.
Finally, to investigate the potential of the MNPs-RBC constructs as MRI contrast agents, free-MNPs and MNPs-RBCs were dispersed in agarose and analyzed using the T2-MSME sequence. For MNPs 29, the obtained R2 was 405 mM−1 s−1, which is very high when compared to reported values in the literature. Once loaded into RBCs, the R2 of these nanoparticles decayed 10-fold, with a value of 40 mM−1 s−1. Concerning the MNPs 40, the measured R2 was lower than those of the corresponding iron oxide nanoparticles, with a value of 260 mM−1 s−1. Anyway, the encapsulation in RBCs did not affect drastically the R2 value, with a 2.3-fold decay at 110 mM−1 s−1. The different behavior is representative of the different encapsulation of these two MNPs, whereas the MNPs 29 tended to produce small aggregates in the RBCs, and the smaller MNPs 40 nanoparticles were found to be more individually dispersed in the cell cytoplasm, as evidenced in Fig. 11.
The systematic study conducted on human and murine erythrocytes has allowed to highlight the peculiar aspects of the synthesis of magnetic nanoparticles for the engineering of these circulating cells. We proposed synthesis protocols to overcome the limitations of non-optimal material that mostly led to the presence of particles in aggregated forms on the extracellular surface at the end of the loading procedure, by developing more suitable nanomaterials. The first requirement definitely concerns the stability of the nanomaterials, which was found related to the dispersity and size of the particles, but also to the surface coating. In this case, a polysaccharidic coating, based on dextran, proved to be excellent in terms of stability and long-term storage. Secondly, MNPs must not affect RBC viability, permitting a cell recovery at the end of the loading procedure similar to that of control cells. Within the different synthetic parameters, filtrations and a fine control of the crystal growth time were the most impactful parameters for the resulting MNPs. It was evidenced that nanoparticles with a crystal growth time below two minutes showed the best encapsulation performance. Furthermore, following the popular strategy for tailoring the MNPs properties of introducing divalent ions such as manganese and zinc into the crystalline structure,38 we showed that the obtained Zn/Mn FNPs exhibited a reduction in nanoparticles size, through a modification of the crystal lattice parameters of the partially doped-spinel structure.39 Unfortunately, the introduction of zinc and manganese resulted in a reduced saturation magnetization (and therefore a reduced R2 relaxivity) but, remarkably, this strategy further enhanced the loading of these ferrites into human red blood cells. Our study allowed to select those nanomaterials that are most suitable for loading into RBCs producing new biocompatible MNP-constructs particularly useful for the imaging of the circulatory system. This takes on even more significance considering that commercial and efficient iron-based contrast agent such as Resovist® (still considered one of the gold standards for imaging techniques as MRI and MPI) is currently available only in Japan. In addition to that, the Gd-based paramagnetic contrast agents that are routinely used alternatively as standard T1-contrast agents for clinical MRI nowadays,40 can be highly toxic leading in some cases to the possible risk of acute renal failure and accumulation in bones, liver and spleen. Recently, safety concerns were raised for this class of agents, since gadolinium chelates are deposited in the brain for months or years, with particularly alarming long-term effects for pediatric diagnostic.41 In this context, the present study permitted to understand how to improve the properties of newly synthesized nanoparticle suspensions to obtain MNPs-RBCs which in the next future we propose to use for imaging with MRI/fMRI. The novelty of this research consists in the production of magnetic nanoparticles suitable for the encapsulation procedure within human and murine RBCs. We consider it as a very important result since not all magnetic nanoparticles (synthesized from other groups in the past) can be encapsulated in the RBCs; moreover, the MNP suspensions here presented are very stable over time (months) and maintain their monodispersed form in the suspensions (MNPs 29 and MNPs 40 also) without precipitation. In addition, the Fe molar concentrations reached resulted in an optimal range to perform the dialysis step with RBCs. This is important to avoid the decrease of the RBC hematocrit percentage which can lead to a lower final cell recovery and therefore a lower yield to be used in vivo. An in vivo study in mice involving the best-performing MNP-RBC has been already set. We could envisage that the preliminary pharmacokinetic results obtained with murine RBCs will lead to a prolongment of MNPs survival in the blood circulation. The absence of nanomaterial on the cell surface membrane (or in the extracellular space) will avoid the fast phagocytosis by RES in the liver of MNPs-loaded-RBCs, as previously observed in in vivo analysis of Ferucarbotran-loaded RBCs.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3bm00264k |
‡ These authors contributed equally to this work. |
This journal is © The Royal Society of Chemistry 2023 |