Science with Passion
Application No.: VPH0079 Version 1 / 03/2024
Co-precipitation of metal carbonate templated protein microparticles –
using the KNAUER IJM NanoScaler
S. Stephan1, N. Elghobashi-Meinhardt1;
stephan@knauer.net
Yu Xiong2, Hans Bäumler2
1KNAUER Wissenschaftliche Geräte GmbH, Hegauer Weg 38,
14163 Berlin;
2Charité-Universitätsmedizin Berlin
3D Rendering: KNAUER
Summary
The KNAUER IJM NanoScaler can be used to precipitate protein metal carbonate hybrid particles by mixing two inorganic salt solutions (MnCl2 and Na2CO3) in which the protein is also dissolved. Adding suitable additives allows the production of such hybrid particles in µm range mainly as single particles with only a small number of aggregates.
Introduction
The Co-precipitation-Crosslinking-Dissolution (CCD) technique allows a few-step-fabrication of particles composed of different biopolymers and bioactive agents under mild conditions without usage of any organic solvents [1, 2]. When mixing two or more inorganic salt solutions (e.g. MnCl2 and Na2CO3) in which the biopolymers are also dissolved, water-insoluble template particles/metal carbonate particles are formed and the biopolymers are encapsulated in the particles during precipitation (co-precipitated hybrid particles) [1]. These hybrid particles are treated with a suitable crosslinker so that the biopolymers encapsulated in the template are crosslinked. After washing, the particles are further treated with a solution that can dissolve the inorganic template (e.g. EDTA, hydrochloric acid). After purification, pure biopolymer particles are obtained without the inorganic salt originally used [1]. Currently, the CCD technique is being implemented to develop hemoglobin and albumin particles that can act as oxygen- or drug-carriers [2–7].
The co-precipitation step is the key process of the CCD technique and largely determines the size and morphology of the biopolymer particles and their yield. The co-precipitation step can be carried out manually in the laboratory by pouring the two inorganic salt solutions together while stirring. However, this method is not standardized. For rigorous product development, it is imperative to implement a standardized process for co-precipitation that complies with "good manufacturing practice" (GMP) and that is scalable and automatable. Here, we investigate whether the traditional co-precipitation step of the CCD method, in which bulk solutions are manually mixed in a beaker, can be replaced by automatized mixing with the Impingement Jets Mixing (IJM) technology of the NanoScaler. The size of the precipitated protein particles should be adjustable in the range of 0.1 to 10 μm.
Sample Preparation
0.25 M MnCl2 containing 10 mg/ml hemoglobin and 0.25 M Na2CO3 were mixed using KNAUER IJM #5. 0.5 mg/ml human serum albumin (HSA) solution was used to dilute/block the precipitation. Different flow rates and additives were tested (Tab. 1). Samples were analyzed by dynamic light scattering (DLS) and confocal laser scanning microscopy (CLSM).
After each experiment, to avoid clogging by remaining solution in the system, the NanoScaler was immediately washed with distilled water, followed by flushing with 0.5 M EDTA pH 7.4, distilled water again and 30 % ethanol.
Tab. 1 Flow rates, additives used, and final concentration of additive
Flow rate (ml/min) | ||
MnCl2-Hb/Na2CO3/HSA | Additive in Na2CO3 solution | Final concentration of additive in precipitate before dilution with HSA |
1/1/3 | – | |
2/2/6 | – | |
2/2/6 | Hyaluronic acid (HA) | 0.1 % |
2/2/6 | Hydroxyethyl starch (HES) | 2 % |
2/2/6 | Oxidized dextran 70 (ODx) | 2 % |
2/2/6 | Dextran 70 (Dx) | 2 % |
2/2/6 | PEG 10kD | 2 % |
1/1/3 | Dextran 70 | 2% |
3/3/9 | Dextran 70 | 2 % |
5/5/15 | Dextran 70 | 2 % |
10/10/30 | Dextran 70 | 2 % |
12/12/30 | Dextran 70 | 2 % |
Results
With a flow rate of 2 ml/min for both MnCl2-Hb and Na2CO3 solutions, followed by dilution with 6 ml/min HSA solution, Hb-MnCO3 hybrid particles with aggregation are produced (Fig. 1).
Fig. 1 CLSM images of Hb-MnCO3 hybrid particles produced with KNAUER IJM #5. Flow rate of MnCl2-Hb/Na2CO3: 2 ml/min, HSA: 6 ml/min. Particles are aggregating.
Adding 0.2–4 % biopolymer (e. g. hyaluronic acid, PEG, dextran) into the Na2CO3 solution caused an increase in size of single particles up to 2.5 µm, but the aggregation of hybrid particles decreased. A 2 % dextran solution added during the precipitation with an increased flow rate (10 ml/min for both MnCl2-Hb and Na2CO3 solutions, 30 ml/min for HSA solution), led to formation of spherical hybrid particles with size range of 1–2 µm and with reduced aggregation, as observed under CLSM (Fig. 2).
Fig. 2 CLSM images of Hb-MnCO3 hybrid particles produced with KNAUER IJM # 5. Flow rate of MnCl2-Hb/dextran-Na2CO3: 10 ml/min, HSA: 30 ml/min. Dextran final concentration before dilution with HSA: 2 %. Particle sizes range between 1–2 µm.
DLS measurements of hybrid particles with dextran as an additive indicate a narrow size distribution by number (Fig. 3).
Size distribution of Hb-MnCO3 hybrid particles produced with IJM #5
Fig. 3 Flow rate of MnCl2-Hb/dextran-Na2CO3: 10 ml/min, HSA: 30 ml/min. Dextran final concentration before dilution with HSA: 2 %
Conclusion
It is possible to precipitate protein-metal carbonate hybrid particles with low aggregation using a KNAUER IJM NanoScaler if suitable additives, such as dextran, are used. Currently, hybrid particles with low aggregation in the µm range can be produced. Other polymers as additives or block solution and IJM mixing chambers of varying size, are currently being tested.
Material and Methods
Substances
Bovine hemoglobin (Actoheme®) was provided by Biophyll GmbH, Dietersburg, Germany. Human serum albumin (Plasbumin®20) was purchased from Grifols Deutschland GmbH, Frankfurt am Main, Germany. Manganese chloride tetrahydrate (MnCl2·4H2O), sodiumcarbonate (Na2CO3), ethylene-diaminetetraacetic acid (EDTA), hyaluronic acid sodium salt, PEG 10kD, sodium hydroxide and sodium (meta) periodate were were purchased from Sigma-Aldrich, Darmstadt, Germany. Ethanol was purchased from Carl Roth GmbH, Karlsruhe, Germany. Ampuwa® (aqua ad injectable) was purchased from Fresenius Kabi Deutschland GmbH, Bad Homburg, Germany. Dextran (from Leuconostoc mesenteroides, M.W. 70kDa) was purchased from AppliChem GmbH (Darmstadt, Germany). Hydroxyethyl starch (HES) 130/0,42 was provided by Serumwerk Bernburg AG, Bernburg, Germany. Oxidized dextran 70KD was synthesized as described previously [4].
Method
Confocal laser scanning microscopy (CLSM)
CLSM images were taken with a confocal microscope LSM 510 Meta (Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with a 100 x oil-immersion objective (numerical aperture 1.3) applying excitation wavelength of 488 nm and a long pass emission filter 505 nm. Magnification: 1000 x.
Dynamic light scattering (DLS)
The hydrodynamic size of hybrid particles was determined using a Zetasizer Nano ZS instrument (Malvern Instruments Ltd., Worcestershire, UK). Each sample was diluted 1:200 using Ampuwa®. All the measurements were carried out in triplicate.
Instrument | Description | Article No. |
NanoScaler | A48040C |
References
[1] Xiong, Y., Georgieva, R., Steffen, A., Smuda, K. & Bäumler, H. Structure and properties of hybrid biopolymer particles fabricated by co-precipitation cross-linking dissolution procedure. J. Colloid Interface Sci. 514, 156–164 (2018).
[2] Xiong, Y. et al. Nonvasoconstrictive Hemoglobin Particles as Oxygen Carriers. ACS Nano 7, 7454–7461 (2013).
[3] Li, H. et al. Photo-Decomposable Sub-Micrometer Albumin Particles Cross-Linked by ortho-Nitrobenzyl Derivatives. Macromol. Chem. Phys. 1–6 (2017).
[4] Kloypan, C. et al. Improved oxygen storage capacity of haemoglobin submicron particles by one-pot formulation. Artif. Cells, Nanomedicine, Biotechnol. 46, S964–S972 (2018).
[5] Li, Y. et al. Functionalized protein microparticles targeting hACE2 as a novel preventive strategy for SARS-CoV-2 infection. Int. J. Pharm. 638, (2023).
[6] Suwannasom, N. et al. Albumin submicron particles with entrapped riboflavin-fabrication and characterization. Nanomaterials 9, (2019).
[7] Chaiwaree, S. et al. Doxorubicin–loaded human serum albumin submicron particles: Preparation, characterization and in vitro cellular uptake. Pharmaceutics 12, 1–15 (2020).
Application details
Method |
LNP |
Mode |
Other |
Substances |
biopolymers, metal carbonate |
Version |
Application No.: VPH0079 Version 1 | 03/2024 | ©KNAUER Wissenschaftliche Geräte GmbH |