This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.
Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) – a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.
Molecular imaging is the non-invasive and targeted visualization of biological processes at the cellular, subcellular, and molecular levels1. Molecular imaging permits a specimen to remain in its native microenvironment while its endogenous pathways and mechanisms are assessed in real-time. Typically, molecular imaging involves the administration of an exogenous imaging agent in the form of a small molecule, macromolecule, or nanoparticle to visualize, target, and trace relevant physiological processes being studied2. The various imaging modalities that have been explored in molecular imaging include MRI, CT, PET, SPECT, ultrasound, photoacoustics, Raman spectroscopy, bioluminescence, fluorescence, and intravital microscopy3. Multimodal imaging is the combination of two or more imaging modalities where the combination enhances the ability to visualize and characterize various biological processes and events4. Multimodal imaging exploits the strengths of the individual imaging techniques, while compensating for their individual limitations3.
This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) – a novel class of multimodal, molecular imaging agents. The PB NPs are utilized for fluorescence imaging and molecular MRI. PB is a pigment consisting of alternating iron (II) and iron (III) atoms in a face-centered cubic network (Figure 1). The PB lattice is comprised of linear cyanide ligands in a FeII– CN – FeIII linkage that incorporates cations to balance charges within its three-dimensional network5. The ability of PB to incorporate cations into its lattice is exploited by separately loading gadolinium and manganese ions into the PB NPs for MRI contrast.
The rationale for pursuing a nanoparticle design for MRI contrast is because of the advantages this design offers relative to current MRI contrast agents. The vast majority of US FDA-approved MRI contrast agents are gadolinium chelates that are paramagnetic in nature and provide positive contrast by the spin-lattice relaxation mechanism6,7,8. As compared to a single gadolinium-chelate that provides low signal intensity on its own, the incorporation of multiple gadolinium ions within the PB lattice of the nanoparticles provides enhanced signal intensity (positive contrast)3,9. Further, the presence of multiple gadolinium ions within the PB lattice increases the overall spin density and the magnitude of paramagnetism of the nanoparticles, which disturbs the local magnetic field in its vicinity, thereby generating negative contrast by the spin-spin relaxation mechanism. Thus the gadolinium-containing nanoparticles function both as T1 (positive) and T2 (negative) contrast agents10,11.
In a subset of patients with impaired renal function, the administration of gadolinium-based contrast agents has been linked to the development of nephrogenic systemic fibrosis8,12, 13. This observation has prompted investigations into the use of alternative paramagnetic ions as contrast agents for MRI. Therefore, the versatile design of the nanoparticles is adapted to incorporate manganese ions within the PB lattice. Similar to gadolinium-chelates, manganese-chelates are also paramagnetic and are typically used to provide positive signal intensity in MRI7,14. As with gadolinium-containing PB NPs, the manganese-containing PB NPs also function as T1 (positive) and T2 (negative) contrast agents.
To incorporate fluorescence imaging capabilities, the nanoparticle “cores” are coated with a “biofunctional” shell consisting of the fluorescently-labeled glycoprotein avidin (Figure 1). Avidin not only enables fluorescence imaging, but also serves as a docking platform for biotinylated ligands that target specific cells and tissue. The avidin–biotin bond is one of the strongest known, non-covalent bonds characterized by extremely strong binding affinity between avidin and biotin15. The attachment of biotinylated ligands to the avidin-coated PB NPs confers molecular targeting capabilities to the PB NPs.
The motivation for pursuing fluorescence and MR imaging using PB NPs is because these imaging modalities possess complementary features. Fluorescence imaging is one of the most widely used optical molecular imaging techniques, and allows for the simultaneous visualization of multiple objects at high sensitivities1,16,17. Fluorescence imaging is a safe, non-invasive modality but is associated with low depths of penetration and spatial resolutions1,3,16. On the other hand, MRI generates high temporal and spatial resolution non-invasively and without a need for ionizing radiation1,3,16. However MRI suffers from low sensitivity. Therefore fluorescence imaging and MRI were selected as the molecular imaging techniques due to their complementary features of depth penetration, sensitivity, and spatial resolution.
This article presents the protocol for the synthesis and biofunctionalization of the PB NPs, gadolinium-containing PB NPs (GdPB), and manganese-containing PB NPs (MnPB)10,11. The following methods are described: 1) measurement of size, charge, and temporal stability of the nanoparticles, 2) evaluation of cytotoxicity of the nanoparticles, 3) measurement of MRI relaxivities, and 4) utilization of the nanoparticles for fluorescence and molecular MR imaging of a population of targeted cells in vitro. These results demonstrate the potential of the NPs for use as multimodal, molecular imaging agents in vivo.
1. Synthesis of PB NPs, GdPB, and MnPB
Synthesis of the nanoparticles (PB NPs, GdPB, or MnPB) is achieved using a one-pot synthesis scheme by performing the steps detailed below:
2. Biofunctionalization of PB NPs, GdPB, and MnPB
Biofunctionalization of the nanoparticles involves coating of the nanoparticle “cores” with avidin and adding biotinylated ligands as described below:
3. Sizing, Zeta Potential, and Temporal Stability of the Nanoparticles
The size distribution, charge, and stability of the nanoparticles are measured using dynamic light scattering (DLS) methods as described below:
4. Cytotoxicity of the Nanoparticles
Cytotoxicity of the nanoparticles is measured using an XTT cell proliferation assay as follows:
5. MRI Relaxivities of the PB NPs, GdPB, and MnPB
MRI relaxivity is measured using T1– and T2-weighted sequences by preparing an MRI “phantom” using a 96-well plate containing nanoparticles as described below:
6. Fluorescent Labeling of Targeted Cells Using the Nanoparticles — Confocal Microscopy
NOTE: The nanoparticles (PB NPs, GdPB, and MnPB) can be used to fluorescently label a population of targeted cells (monitored by confocal microscopy) as follows:
7. Fluorescent Labeling of Targeted Cells Using the Nanoparticles — Flow Cytometry
The nanoparticles (PB NPs, GdPB, and MnPB) can be used to fluorescently label a population of targeted cells (monitored by flow cytometry) as follows:
8. Generating MRI Contrast on Targeted Cells Using the Nanoparticles
The nanoparticles (PB NPs, GdPB, and MnPB) can be used to generate MRI contrast (in both T1– and T2-weighted sequences) in a population of targeted cells as follows:
Using the one-pot synthesis scheme, nanoparticles of PB NPs (mean diameter 78.8 nm, polydispersity index (PDI) = 0.230; calculated by the dynamic light scattering instrument), GdPB (mean diameter 164.2 nm, PDI = 0.102), or MnPB (mean diameter 122.4 nm, PDI = 0.124) that are monodisperse (as measured by DLS) can be consistently synthesized (Figure 2A). The measured zeta potentials of the synthesized nanoparticles are less than -30 mV (Figure 2B), indicating moderate stability of the particles based on their surface charges. The synthesized nanoparticles exhibit adequate temporal stability over a period of 5 days as indicated by their consistent sizes (hydrodynamic diameters; Figure 2C).
When co-incubated with cells, the nanoparticles (PB NPs, GdPB, and MnPB) exhibit negligible cytotoxicity to the cells below certain threshold concentrations (Figure 3). Cytotoxicity studies of PB NPs on Neuro2a cells indicate negligible cytotoxicity when co-incubated with Neuro2a at concentrations lower than 0.67 × 10-6 mg/cell (Figure 3A). Cytotoxicity studies conducted by co-incubation of GdPB with EoL-1 and OE21 cells indicate negligible cytotoxicity of GdPB on both cell types at concentrations lower than 0.25 × 10-6 mg/cell (Figure 3B). Similar, cytotoxicity studies indicate negligible cytotoxicity of MnPB when co-incubated with BSG D10 at concentrations lower than 0.25 × 10-6 mg/cell (Figure 3C). The additional cytotoxicity of MnPB and GdPB can be attributed to the presence of additional ions (Mn2+ for MnPB and Gd3+ for GdPB) within the nanoparticle core.
MRI relaxivity studies were conducted using phantoms comprised of varying concentrations of PB NPs, GdPB, and MnPB. The studies indicate the utility of the nanoparticles as MRI contrast agents in both T1W and T2W sequences (Figure 4). This is demonstrated by the increased hyperintensities (positive contrast) in T1-weighted sequences and increased hypointensities (negative contrast) in T2-weighted sequences with increasing concentrations of both GdPB (Figure 4A) and MnPB (Figure 4B). Based on the relaxivity measurements, MnPB is a moderate T1 agent and a strong T2 agent while GdPB is a strong T1 agent and a moderate T2 agent (Figure 4C). The measured relaxivities of GdPB and MnPB compare favorably with those of clinically approved contrast agents10, 11.
The biofunctionalized PB NPs are able to fluorescently label a population of targeted cells in vitro (Figure 5). When biofunctionalized with both fluorescent avidin (A488) and biotinylated anti-human eotaxin-3 antibody (Eot3), GdPB can fluorescently target a population of EoL-1 cells (Figure 5B). Control GdPB nanoparticles without Eot3 exhibit negligible binding (Figure 5A) Similarly, when GdPB is biofunctionalized with both fluorescent avidin (A488) and biotinylated anti-neuronal glial antigen-2 (ANG2), the nanoparticles can fluorescently target populations of BSG D10 and SUDIPG1 neurospheres (Figures 5D and F), while control nanoparticles without the targeting antibody are unable to fluorescently label the cells (Figures 5C and E). Thus, efficient targeting and fluorescent labeling require the presence of both fluorescent avidin and the biotinylated targeting ligand.
The capability of the biofunctionalized nanoparticles to fluorescently label a population of cells, as quantified by flow cytometry, confirms the need for both fluorescent avidin and biotinylated-targeting ligand for effective fluorescent labeling (Figure 6). BSG D10 cells contacted with MnPB containing both A488 and ANG2 (MnPB-A488-ANG2) exhibit increased fluorescence (Figure 6A) and percentage of fluorescently-labeled cells (Figure 6B) when compared to control fluorescent nanoparticles with a control antibody (MnPB-A488-AbC) and without an antibody (MnPB-A488). The biofunctionalized nanoparticles are able to fluorescently target a specific sub-population of cells within a cell mixture (Figure 6C). This is demonstrated as fixed amounts of EoL-1-targeting, fluorescent GdPB (GdPB-A488-Eot3) are contacted with targeted cells (EoL-1) and control cells (OE21). Fluorescence increases as proportions of EoL-1 are increased (increased Alexa Fluor 488 signal intensity; Figure 6C) within the mixture. This indicates the specificity of the nanoparticles for this sub-population of cells within the cell mixture.
The biofunctionalized PB NPs increase MRI contrast in a population of targeted cells (Figure 7). When BSG D10 cells are contacted with equivalent concentrations of experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, phantoms exhibit increased hyperintensity for cells contacted with MnPB-A488-ANG2 compared to controls in T1W sequences, and increased hypointensity for cells contacted with MnPB-A488-ANG2 compared to controls in T2W sequences (Figure 7A). Image analysis of the ROIs within the phantom confirm this trend where cells contacted with experimental particles exhibit significantly increased intensity when compared to controls in T1W sequences and significantly decreased intensity relative to controls in T2W sequences (Figure 7B).
Figure 1: The core-shell design of the PB NPs. The core consists of a PB lattice, which is comprised of linear cyanide ligands in a FeII– CN – FeIII linkage. These linkages enable PB NPs to incorporate cations within its three-dimensional network as a means of balancing charges5. This cation-binding ability of Prussian blue is utilized to load gadolinium and manganese ions within the lattice, which provides MRI contrast. The core is coated with a biofunctional shell comprised of fluorescent avidin to enable fluorescence imaging and biotinylated ligands to enable molecular targeting.
Figure 2: Size, charge, and stability of the PB NPs. (A) Size distributions of PB (blue), GdPB (red) and MnPB (black) nanoparticles measured by DLS. (B) Zeta potentials of PB NPs, GdPB, and MnPB. (C) Temporal stability of PB (blue), GdPB (red) and MnPB (black) nanoparticles in water (solid) and DMEM (dotted) for five days after their synthesis, measured by DLS.
Figure 3: Cytotoxicity of the PB NPs. Cytotoxicity studies using various concentrations of (A) PB NPs added to a fixed number of Neuro2a cells (B) GdPB added to a fixed amount of EoL-1 and OE-21 cells, respectively and (C) MnPB added to a fixed number of BSG D10 cells. Cell survival rate was calculated at 24 and 48 hr. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society; Ref 10 with permission from Dove Press Ltd; and Ref 21 with permission from The Royal Society of Chemistry.
Figure 4: MR images and relaxivities of GdPB and MnPB at 3 T. Hyperintensity in T1-weighted sequences and hypointensity in T2-weighted sequences of (A) GdPB and (B) MnPB as a function of concentration of the nanoparticles. (C) Tabulation of the relaxitivies of PB NPs, GdPB, and MnPB measured at 3 T. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society.
Figure 5: Fluorescent labeling of targeted cells using the biofunctionalized PB NPs, as measured by laser scanning confocal microscopy. Images of EoL-1 cells treated with (A) control (GdPB-A488) and (B) experimental (GdPB-A488-Eot3) nanoparticles. Images of BSG neurospheres treated with (C) control (MnPB-A488) and (D) experimental (MnPB-A488-ANG2) nanoparticles. Images of SUDIPG1 neurospheres treated with (E) control (MnPB-A488) and (F) experimental (MnPB-A488-ANG2) nanoparticles. Reproduced with permission from Ref 11, copyright 2014 American Chemical Society. Please click here to view a larger version of this figure.
Figure 6: Fluorescent labeling of targeted cells using the biofunctionalized PB NPs, as measured by flow cytometry. (A) Flow cytometry histograms of BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles. (B) Percentage of BSG D10 cells from panel (A) that are fluorescent (% of Alexa-Fluor positive) upon treatment with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, ** p <0.05. (C) Flow cytometry scatter plots of a cell mixture containing varying proportions of EoL-1 (targeted cells) and OE21 (control cells) targeted by the nanoparticles (GdPB-A488-Eot3). Reproduced with permission from Ref 11, copyright 2014 American Chemical Society and from Ref 10 with permission from Dove Press Ltd. Please click here to view a larger version of this figure.
Figure 7: Increasing MRI contrast in targeted cells using the biofunctionalized PB NPs. (A) T1-weighted and T2-weighted contrast enhancement in phantoms comprised of a fixed number of BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles. (B) Normalized signal intensity for BSG D10 cells treated with experimental (MnPB-A488-ANG2) and control (MnPB-A488-AbC and MnPB-A488) nanoparticles, ** p <0.05. Reproduced from Ref 10 with permission from Dove Press Ltd.
This article has presented the methods for the synthesis of a novel class of multimodal, molecular imaging agents based on biofunctionalized Prussian blue nanoparticles. The molecular imaging modalities incorporated into the nanoparticles are fluorescence imaging and molecular MRI, due to their complementary features. The biofunctionalized Prussian blue nanoparticles have a core-shell design. The key steps in the synthesis of these nanoparticles are the: 1) one-pot synthesis which yields the cores that are comprised of Prussian blue nanoparticles (PB NPs), gadolinium-containing Prussian blue nanoparticles (GdPB), or manganese-containing Prussian blue nanoparticles (MnPB), 2) biofunctionalization of the nanoparticles using fluorescent avidin by electrostatic self-assembly, and 3) attachment of the biotinylated ligands (antibodies) to the nanoparticles using robust avidin-biotin interactions. Both fluorescent avidin and the biotinylated ligands constitute the biofunctional shell of the nanoparticles.
The one-pot synthesis yields PB NPs, GdPB, or MnPB that function both as T1 and T2 contrast agents for MRI. The amounts of the paramagnetic ions (gadolinium or manganese) loaded into the nanoparticle core can be altered (increased or decreased) by varying the amounts of the paramagnetic ion-containing salts (gadolinium (III) nitrate and manganese (II) chloride) in the one-pot synthesis (Step 1.2). This results in altered (increased or decreased) MRI signal intensities. However, these modifications to the synthesis scheme may result in unstable, aggregated nanoparticles. To mitigate concerns associated with aggregation, the one-pot synthesis may be modified to incorporate size-controlling capping agents such as citrate during synthesis20.
Biofunctionalization of the nanoparticle cores is achieved by electrostatic self-assembly with fluorescent avidin, which enables fluorescence imaging. Electrostatic self-assembly requires opposite charges on the surface of the nanoparticles and the coating polymer (in this article, fluorescent avidin). To alter the surface functionality of the nanoparticles, avidin may be replaced by positively-charged polymers (e.g. polylysine or an amine-group containing polyethylene glycol) during the synthesis. However the relative proportions of the nanoparticles and coating polymer will have to be optimized to maintain nanoparticle size and stability and to prevent aggregation.
Addition of the biotinylated antibodies onto the avidin coated nanoparticles confers molecular targeting capabilities to the PB-based nanoparticles. This step is based on the robust interaction between avidin and biotin (equilibrium dissociation constant, Kd ~10-15). As with previous steps, the relative proportions of the avidin-coated nanoparticles and biotinylated ligands need to be optimized so as to prevent the biotinylated ligand from simultaneously binding two avidin coated nanoparticles resulting in nanoparticle aggregation. For molecular targeting, the antibodies may be replaced by other targeting ligands such as antibody fragments (Fab), single-chain variable fragment (scFv), peptides, or aptamers.
The key advantages of this method for synthesizing biofunctionalized nanoparticles as multimodal, molecular imaging agents are the: 1) facile one-pot (1-step) synthesis of the nanoparticle cores, and 2) sequential contacting steps (electrostatic self-assembly and avidin-biotin interactions) for coating the nanoparticle core with a biofunctional shell. Other advantages of the nanoparticles include the fact that Prussian blue (sold as Radiogardase) is already FDA-approved for human use and that the nanoparticles that results from the one-pot synthesis scheme can be used as an MRI contrast agent both in T1 (positive) and T2 (negative)-weighted sequences, which is not easily achieved using other contrast agents or nanoparticle platforms without complicated synthesis schemes or specialized chemicals for synthesis of the contrast agents. A limitation of the technique is that the synthesis can lead to polydisperse nanoparticles with aggregates if the relative proportions of the reactants in both nanoparticle core synthesis and biofunctional shell coating steps are not rigorously optimized for the reactants used in those particular steps. For example, the relative proportions for coating the nanoparticles cores with avidin cannot be extended to coating the nanoparticles with polylysine without prior optimization studies.
After mastering the technique for synthesizing biofunctionalized Prussian blue nanoparticles described here, this versatile design can be modified for molecular imaging studies in vivo. This will require PEGylation of the nanoparticles for lower immunogenicity and longer circulation times in vivo. Similar to the design described here, the PB NPs can be biofunctionalized with antibodies prior to in vivo administration. Other studies include the use of the biofunctionalized PB NPs for theranostic (simultaneous therapy + diagnostic) applications in vivo. Studies investigating the use of Prussian blue nanoparticles for photothermal therapy (based on their absorbance characteristics at near infrared wavelengths) are currently underway21.
The authors have nothing to disclose.
This work was supported by the Sheikh Zayed Institute for Pediatric Surgical Innovation (RAC Awards #30000174 and 30001489).
Name of Material/ Equipment | Company | Catalog Number | Comments/Description |
Potassium hexacyanoferrate (II) trihydrate (K4Fe(CN)6 · 3H2O) | Sigma-Aldrich | P9387 | |
Manganese (II) chloride tetrahydrate (MnCl2 · 4H2O) | Sigma-Aldrich | 221279 | |
Gadolinium (III) nitrate hexahydrate (Gd(NO3)3 · 6H2O) | Sigma-Aldrich | 211591 | |
Iron (III) chloride hexahydrate (FeCl3 · 6H2O) | Sigma-Aldrich | 236489 | |
Sodium chloride (NaCl) | Sigma-Aldrich | S9888 | |
Anti-NG2 Chondroitin Sulfate Proteoglycan, Biotin Conjugate Antibody | Millipore | AB5320 | |
Biotinylated Anti-Human Eotaxin-3 | Peprotech | 500-P156GBT | |
Neuro-2a Cell Line | ATCC | CCL-131 | |
BSG D10 Cell Line | Lab stock | — | |
OE21 Cell Line | Sigma-Aldrich | 96062201 | |
SUDIPG1 Neurospheres | Lab stock | — | |
Eol-1 Cell Line | Sigma-Aldrich | 94042252 | |
Poly(L-lysine) hydrobromide | Sigma-Aldrich | P1399 | |
Formaldehyde | Sigma-Aldrich | F8775 | |
Bovine serum albumin | Sigma-Aldrich | A2153 | |
Aminoactinomycin D | Sigma-Aldrich | A9400 | |
Triton X-100 | Sigma-Aldrich | X100 | |
CellTrace Calcein Red-Orange, AM | Life Technologies | C34851 | |
Avidin-Alexa Fluor 488 | Life Technologies | A21370 | |
Centrifuge | Eppendorf | 5424 | |
Peristaltic Pump | Instech | P270 | |
Zetasizer Nano ZS | Malvern | ZEN3600 | |
Sonicator | QSonica | Q125 | |
Hot Plate/Magnetic Stirrer | VWR | 97042-642 | |
Ultra Clean Aluminum Foil | VWR | 89107-732 | |
Vortex Mixer | VWR | 58816-121 | |
1.7 mL conical microcentrifuge tubes | VWR | 87003-295 | |
15 mL conical centrifuge tubes | VWR | 21008-918 | |
Tube holders | VWR | 82024-342 | |
Disposable plastic cuvettes | VWR | 7000-590 (/586) | |
Zetasizer capillary cell | VWR | DTS1070 | |
Centrifugal Filters, 0.2 micrometer spin column | VWR | 82031-356 | |
96-well cell culture tray | VWR | 29442-056 | |
Trypsin EDTA 0.25% solution 1X | JR Scientific | 82702 | |
Cell Culture Grade PBS (1X) | Life Technologies | 10010023 | |
XTT Cell Proliferation Assay Kit | Trevigen | 4891-025-K | |
T75 Flask | 89092-700 | VWR | |
Dulbecco's Modified Eagle's Medium | Biowhitaker | 12-604Q | |
Fetal Bovine Serum | Life Technologies | 10437-010 | |
Pen-Strep 1X | Life Technologies | 15070063 | |
Fluoview FV1200 Confocal Laser Scanning Microscope | Olympus | FV1200 | |
Chambered Microscope Slides | Thermo Scientific | 154534 | |
Micro Cover Glasses, Square, No. 1.5 | VWR | 48366-227 | |
Microscope Slides | VWR | 16004-368 | |
RPMI | Sigma-Aldrich | R8758 | |
Agarose | Sigma-Aldrich | A9539 | |
FACSCalibur Flow Cytometer | BD Biosciences | ||
3 T Clinical MRI Magnet | GE Healthcare | ||
100 mL round-bottom flask |