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Abstract

Transfection agent (TFA)-induced magnetic cell labeling with Feridex IV is an attractive method of loading cells because it employs a pharmaceutical source of iron oxide. Although attractive, the method has two significant drawbacks. First, it requires mixing positively charged transfection agents and negatively charged magnetic nanoparticles, and the resulting loss of nanoparticle surface charge causes nanoparticle precipitation. Second, it can result in nanoparticle adsorption to the cell surface rather than internalization. Internalization of Feridex (and associated dextran) is important since dextran cell exterior can react with the antidextran antibodies, commonly present in human populations, and trigger an antibody-mediated cytotoxicity. Here we employed three assays for selecting Feridex/TFA mixtures to minimize nanoparticle precipitation and surface adsorption: (1) an assay for precipitation or stability (light scattering), (2) an assay for labeled cells (percentage of cells retained by a magnetic filter), and (3) an antidextran-based assay for nanoparticle internalization. Cells loaded with Feridex/protamine had internalized iron, whereas cells loaded with Feridex/Lipofectamine had surface-adsorbed iron. Optimal conditions for loading cells were 10 mg/Feridex and 3 mg/mL protamine sulfate. Conditions for loading cells with Feridex and a TFA need to be carefully selected to minimize nanoparticle precipitation and dextran adsorption to the cell surface.

LOADING CELLS WITH MAGNETIC NANOPARTICLES and tracking their fate by magnetic resonance imaging (MRI) in vivo is an attractive approach for enhancing the efficacy of novel cell-based therapies, including those using hematopoietic stem cells,¹ neuroprogenitor cells,^2,3 and immune-specific T cells.⁴ The use of Feridex IV nanoparticles in conjunction with a transfection agent (TFA) (internalization enhancing agent) to increase the uptake by a cell has been widely employed because of the availability of materials and possible low barriers to clinical use.^5–7

A significant issue that arises when cells are exposed to Feridex in vitro is whether the nanoparticle is internalized or adsorbed on the surface. The issue arises because (1) Feridex is a highly polydisperse mixture (contains multiple sizes) of dextran nanoparticles, which can have different propensities for internalization or adsorption;^8,9 (2) studies of the mechanism of the adverse reactions to dextran indicate a high percentage of individuals with antidextran antibodies, some to a very high levels;^10,11 and (3) cells loaded with Feridex/TFA mixtures in which the nanoparticle is surface adsorbed will likely react with antidextran antibodies and be eliminated by antibody-mediated cytotoxicity, instead of serving their desired therapeutic function, which requires long-term cell viability in the host.

We have observed that significant amounts of Feridex become cell associated without the use of TFAs (Feridex at 100 mg Fe/mL) and at temperatures below 4°C, conditions that favor surface adsorption over internalization.¹² In vitro assays used to date for the effects of Feridex loading on cells include assays for cell death, apoptosis, function, and differentiation.^5,13–15 These assays, although indicating a lack of toxicity of Feridex loading over the time course of the assay, provide no information on the intracellular disposition of iron or the propensity for attack by the host immune system. Typically, the intracellular disposition of Feridex has been inferred from microscopy. However, the spatial resolution of most microscopic methods cannot distinguish dextran nanoparticles adsorbed to the outer plane of the membrane, where they can react with antidextran antibodies and trigger a cytotoxic response, from those at or near the inner plane of the membrane, where they would be shielded from antibodies. We previously judged that the Lipofectamine 2000 (LFA)-assisted Feridex loading of C17.2 cells yielded an internalized form of iron based on microscopy (see Figure 3 of Montet-Abou and colleagues¹²). However, here we show that cells treated with a mixture of Feridex and LFA (mixtures are denoted as Feridex/LFA) react with an antidextran antibody. We therefore conclude that LFA increases cell-associated iron by increasing the adsorption of dextran nanoparticles to the cell surface and that microscopy is not a valid method for determining surface-adsorbed Feridex.

We describe three methods for selecting conditions of Feridex and TFAs to achieve cell labeling and to achieve that labeling without the surface adsorption of nanoparticles. The three methods are summarized in Figure 1. First, we employed an assay for aggregates formed when Feridex and TFAs are mixed.¹² The assay, which can rapidly evaluate stability over a wide range of conditions, distinguishes stable aggregates of Feridex nanoparticles and TFAs (size 200–2,000 nm by light scattering) from larger precipitates. Larger precipitating complexes settle onto and bind adherent cells and cannot be removed by washing. With suspended cells, these complexes precipitate with cells when cells are harvested by centrifugation. Conditions for avoiding precipitation were noted, however. These were (1) low concentrations of both the Feridex and TFA, (2) high concentrations of Feridex with low concentrations of TFA, and (3) high concentrations of TFA with low concentrations of Feridex. Second, we introduce here an assay for determining the percentage of Feridex-loaded cells in a mixture by their retention on a magnetic separator, which allows determination of the percentages of magnetically labeled and unlabeled cells. As an analytic method, the assay provides information about the uniformity of magnetic labeling; it can also be used to eliminate unlabeled cells from a population on a larger scale. Third, we introduce here an assay that distinguishes surface adsorbed from internalized Feridex nanoparticles, termed the permeabilization shift assay. The assay uses a fluoresceinated antidextran antibody whose presence on cells is quantified using fluorescence-activated cell sorter (FACS). The fluorescence of cells that have internalized dextran nanoparticles of Feridex is permeabilization dependent, whereas that of cells with surface-adsorbed nanoparticles is permeabilization independent. Using these three methods, we describe optimal conditions for the TFA-assisted loading of cells with Feridex nanoparticles.

Figure 1.

Selection of conditions for Feridex/transfection agent (TFA)-induced magnetic nanoparticle cell labeling. A, Screening Feridex/TFA mixtures for aggregate formation by light scattering and precipitate formation. B, Screening Feridex/TFA mixtures for uniform cell loading by a magnetic retention assay. Feridex/TFA mixtures are applied to adherent cells, detached with trypsin, and filtered, and the percentages of magnetically retained and nonretained cells are determined. C, Screening Feridex/TFA by a permeabilization shift assay. When dextran nanoparticles are adsorbed to the surface, fluoresceinated antidextran binds with or without permeabilization. When nanoparticles are internalized by cells, permeabilization is required for dextran recognition.

Materials and Methods

Materials

Feridex IV served as a source of magnetic nanoparticles. Feridex IV is a registered trademark of Advanced Magnetics (Advanced Magnetics, Cambridge, MA) and is referred to as Feridex. The internalizing enhancing TFAs were protamine sulfate (ProS), the injectable drug (American Pharmaceutical Partners, Los Angeles, CA), and Lipofectamine 2000 (LFA) from Invitrogen (Carlsbad, CA). Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle (MEME), fetal bovine serum (FBS), horse serum (HS), and Hank's Balanced Salt Solution (HBSS) were from Mediatech, Cellgro (Herndon, VA). OptiMemI, L-glutamine, sodium pyruvate, penicillin-streptomycin, and phosphate-buffered saline (PBS) were from Gibco-Invitrogen. The C17.2 cells were from Dr. Evan Snyder; the H9c2, BT-20, and HeLa cell lines were obtained from American Tissue Culture Collection (Manassas, VA).

Cell Culture

C17.2 cells, a mouse neuroprogenitor cell line, were cultured in DMEM supplemented with 10% FBS, 5% HS, 2 mM l-glutamine, sodium pyruvate, and 1 unit of penicillin-streptomycin at 37°C and 5% CO₂. H9c2 cells, a rat embryonic myoblastic cardiac cell line, were cultured in DMEM supplemented with 10% FBS, 5% HS, 2 mM l-glutamine, and 1 unit of penicillin-streptomycin at 37°C and 5% CO₂. BT-20 and HeLa cells were cultured in MEME with 2 mM L-glutamine and Earle salt solution adjusted to contain 1.5 g/L sodium bicarbonate, 0.1 mM nonessential amino acids and 1.0 mM sodium pyruvate, and 10% FBS at 37°C in a humidified 5% CO₂ atmosphere.

TFA-Assisted Feridex Loading

Cells were plated in a six-well plate, grown to 90% confluence, and washed three times with HBSS. They were then incubated (2 hours at 37°C) with the TFA and Feridex in the media used for growth (without antibiotics). Preparation of the Feridex/LFA was first made in OptiMemI (a serum-reduced media) to allow iron and LFA interactions as recommended by the manufacturer.

Feridex/TFA Stability Assay

Incubations of TFAs with Feridex were in PBS for 2 hours at 37°C. The intensity-weighted diameter (size in nm) was determined by dynamic light scattering with a Zetasizer HS1000 (Malvern, Marlboro, MA).

Magnetic Cell Retention Assay

After loading with Feridex and TFA, cells were washed three times with HBSS, harvested by trypsinization, and resuspended in MACS buffer (0.5% BSA, 2 mM ethylenediaminetetraacetic acid, PBS). They were passed through a 35 mm filter, and cell numbers were determined by a hemocytometer. Cells were then applied to a magnetic column (MACS LS Column, Miltenyi, Auburn, CA) held by a magnet (MidiMACS separator, Miltenyi), yielding retained and nonretained cells whose numbers were determined.

Permeabilization Shift Assay

Retained and nonretained cells were divided into two samples, one of which was permeabilized (denoted P), using a commercial kit (Cytofix/Cytoperm kit, BD Biosciences Pharmingen, San Diego, CA), and a second, which was not permeabilized (denoted NP). Each sample was then incubated with a fluoresceinated antidextran (Fl-antidextran, Stem Cell Technologies, Seattle, WA) diluted fivefold (in MACS buffer for nonpermeabilized cells and in the permeabilization/wash buffer for permeabilized cells) for 30 minutes at 4°C. All samples of cells were then washed and resuspended in MACS buffer. Cell-associated antidextran was determined from values of the median fluorescence obtained with the FACScalibur (Becton Dickinson, Franklin Lakes, NJ). The proportion of surface-adsorbed nanoparticles was calculated as the fluorescence of the intact cells divided by the fluorescence of permeabilized cells. Here intact cells exhibit fluorescence from surface-adsorbed nanoparticles, whereas permeabilized cells exhibit fluorescence from both surface-adsorbed and internalized nanoparticles.

Microscopy (Immunohistochemistry)

Cells were grown to ≈90% confluence on chamber slides (Lab-tek II, Nalgenunc Int., Naperville, IL), exposed to Feridex and TFA as above, and washed three times with HBSS. Cells were then fixed with acetone (4°C, 5 minutes) or with paraformaldehyde (room temperature, 20 minutes) and permeabilized by treatment with 0.2% Triton X-100 (room temperature, 5 minutes). All samples were then washed with PBS and incubated for 1 hour at room temperature with the antidextran–fluorescein isothiocyanate antibody diluted sixfold in PBS with sodium azide (0.5%), H₂O₂ (0.3%), and rabbit serum (10%). Slides were washed three times with PBS and exposed to an antifluorescein–horseradish peroxidase conjugate (Invitrogen) diluted to 5 μg/mL (PBS with 2% serum from the same species as the cells) for 40 minutes at room temperature. After three washes in PBS, enzyme activity was developed using AEC substrate (DAKO, Carpinteria, CA). The samples were counterstained with hematoxylin, mounted with mounting medium, and analyzed under the microscope (Nikon E400).

Microscopy (Fluorescent)

After the FACS analysis, the remaining cells were dried on slides and stored at 280°C. After thawing, slides were washed twice with PBS and were incubated with fluorescent stains: nuclei were stained with Hoechst (nuclear stain #33342) and membranes were stained with Alexafluor 594 (wheat germ agglutinin) (Molecular Probes Inc., Invitrogen, Eugene, OR). After three additional washes with PBS, the slides were observed under an epifluorescence microscope (Nikon E80i).

Results

Six Feridex/TFA mixtures were evaluated for stability (Table 1), and five combinations were selected for application to C17.2 cells (Figure 2A). The data in Table 1 are adapted from Montet-Abou and colleagues,¹² in which the stability of nanoparticle/TFA mixtures over a wide range of conditions (temperatures, types of nanoparticles, wide range of concentrations) was evaluated. Evaluation of mixtures for stability is performed first because cells are not needed.

C17.2 cells, loaded when adherent, were then harvested by trypsinization and analyzed by the magnetic cell retention assay, as shown in Figure 2A. The magnetic retention assay indicated that all five Feridex/TFA mixtures resulted in high ratios of retained to nonretained cells, that is, uniform cell labeling with iron, and provided no basis for selecting the conditions for cell loading.

Magnetically retained cells from the five Feridex/TFA mixtures, selected for stability and magnetic retention, were then split into two portions, one of which was permeabilized and both of which were reacted with Fl-antidextran. As shown by the FACS analysis in Figure 2B, cells treated with Feridex/ProS (10 μg/mL Feridex, 3 μg/mL ProS) did not bind Fl-antidextran unless permeabilized, indicating that the dextran nanoparticle had been internalized. However, cells treated with Feridex/LFA (10 μg/mL Fe, 3 μg/mL LFA) bound Fl-antidextran with or without permeabilization, indicating that the nanoparticle was on the cell surface and had not been internalized. We term the increased binding of Fl-antidextran with permeabilization a “permeabilization shift assay.” The results of the permeabilization shift assay with all five Feridex/TFA mixtures are shown in Figure 2C.

Table 1.

Stability of Feridex/Transfection Agent Mixtures

Feridex (μgFe/mL)	0 μg/mL ProS	1 μg/mL ProS	3 μg/mL ProS
10	167	2,314	2,180
30	173	Ppt	Ppt
100	167	1,275	1,620
	0 μg/mL LFA	1.0 μg/mL LFA	3.0 μg/mL LFA
0	NA	410	623
10	167	1,760	1,198
30	169	157	1,434
100	172	293	152

LFA = Lipofectamine; NA = not available: Ppt = precipitate; ProS = protamine sulfate.

The ability of mixtures of Feridex and transfection agents to form stable aggregates rather than precipitates is defined as stability. Numbers are diameters in nanometers.

The Fl-antidextran antibody bound Feridex and not structures normally present on cells. This is evident from controls in which cells (permeabilized or unpermeabilized) treated with ProS or LFA were exposed to antibody and binding was not seen (see Figure 2C).

To confirm the results of the FACS-based permeability shift assay, which quantitates Fl-antidextran binding to cells after detachment by trypsinization, we examined the binding of Fl-antidextran to adherent C17.2 cells using an antifluorescein–horseradish peroxidase conjugate to detect bound Fl-antidextran (Figure 3A). Cells were treated with Feridex/ProS (10 μg/mL Feridex and 3 μg/mL ProS) and were permeabilized or nonpermeabilized. A small amount of Fl-antidextran binding occurred without permeabilization, but binding was greatly increased by permeabilization. The specificity of the antidextran antibody was again shown; control cells not treated with Feridex did not bind the Fl-antidextran antibody. Cells had large blue nuclei with several darker nucleoli per nuclei and relatively little cytoplasm.

Triple-channel fluorescence microscopy was performed (Figure 3B) on the same population of cells (permeabilized or nonpermeabilized) analyzed by FACS. We used a nuclear stain (Hoescht 33342, blue), a membrane-targeted stain (Alexafluor 594 [wheat germ agglutinin], red), and visualized Fl-antidextran (green). The binding of Fl-antidextran was not observed unless the cells were permeabilized. Therefore, the increased binding of antidextran antibody obtained with permeabilization and seen with the quantitative FACS method (see Figure 2C) was evident with two qualitative microscopy-based methods (see Figure 3). The intracellular distribution of the antibody (bound to dextran nanoparticles) appeared to be largely non-nuclear with both immunohistochemical and fluorescent microscopy images. However, it was not possible to determine whether the dextran nanoparticle was adsorbed to the membrane or internalized and within the cytoplasm.

We then ran the magnetic retention and permeabilization shift assays, with the indicated concentrations of Feridex and ProS changing from C17.2 cells to H9c2, BT20, and HeLa cells, as shown in Figure 4. Increasing the concentration of Feridex from10 μg Fe/mL to 100 μg Fe/mL at 3 μg/mL ProS was associated with a substantial increase in the proportion of iron that was surface adsorbed, compared with total cell-associated iron. For example, with H9c2 cells, permeabilized cell fluorescence increased from 1.07-fold, whereas the percentage of membrane-adsorbed nanoparticle increased from 1.9-fold. The best conditions for labeling cells and achieving internalization of the dextran nanoparticle were Feridex at 10 μgFe/mL and ProS at 3 μg/mL. With C17.2 cells, surface adsorption of dextran nanoparticles was undetectable, whereas with H9c2, BT-20, and HeLa cells, surface-adsorbed nanoparticles were less than 25% of total cell-associated nanoparticles.

Figure 2.

Permeabilization shift assay for nanoparticle internalization with C17.2 cells. Five mixtures giving stable solutions (see Table 1) were used. A, Magnetic retention assay for uniform loading of cells. Retained cells were analyzed by permeabilization shift assay. B, Fluorescence-activated cell sorter analysis of cells loaded as indicated, both nonpermeabilized and permeabilized, were reacted with fluoresceinated antidextran (Fl-antidextran). Although Feridex (Fe)/lipofectamine (LFA)-treated cells were magnetically retained, they reacted with Fl-antidextran without permeabilization. Fe/protamine sulfate (ProS)-treated cells do not react with antidextran unless permeabilized. C, Permeabilization shift assay for internalization. White and black bars are median fluorescence without or with permeabilization (NP, P). Feridex (10 μg Fe/mL) with ProS (3 μg/mL) gives permeabilization-dependent labeling of magnetically retained cells, demonstrating internalization. LFA gives surface-based adherence of Feridex, which reacts with antidextran without permeabilization. Cells treated with transfecting agents, but not Feridex, and not retained by the magnetic filter did not react with Fl-antidextran, with or without permeabilization.

Discussion

Each of the three assays shown in Figure 1 evaluates different aspects of the Feridex/TFA mixtures and can be used to maximize cell-associated iron while minimizing surface adsorption of dextran nanoparticles. The Feridex/stability assay, a non–cell-based test for solution stability, was employed to define conditions in which precipitates of Feridex and TFAs do not form. We previously evaluated Feridex mixtures with three TFAs, poly-l-lysine, LFA, and ProS, and found precipitate formation over a wide range of concentrations of Feridex and TFA, which causes the problems outlined above. Thus, the Feridex stability assay provides important guidance as to mixtures of Feridex and a TFA to avoid using when labeling cells.

Figure 3.

Micrographs of cells after treatment with Feridex (Fe) and protamine sulfate (ProS). A, Adherent C17.2 cells were treated with 10 μg/mL Fe and 3 μg/mL ProS. Cells were then permeabilized or left unpermeabilized and exposed to fluoresceinated antidextran (Fl-antidextran), which was visualized with an antifluorescein–horseradish peroxidase conjugate. The micrograph is therefore of adherent cells (×40 original magnification). B, Adherent cells were treated with Fe and ProS as in A. Cells were detached, filtered, purified by magnetic filtration, permeabilized, and reacted with Fl-antidextran. Difference in stain with permeabilization shows internalization of dextran nanoparticles with both techniques. Note the difficulty of distinguishing internalized or surface-adsorbed antibody in all cases (×40 original magnification). Ctl = control.

The magnetic cell retention assay can be used as an analytic method to determine the minimum concentrations of Feridex and TFA needed for producing a population of cells in which all cells in the mixture are magnetically labeled. It can also be used as a preparative method to ensure that the entire cell population is iron loaded even when low concentrations of Feridex and TFA are employed to minimize nonspecific adherence to cells.

The permeabilization shift assay provides a method of assessing whether the dextran nanoparticle is surface adsorbed or internalized. The method employs a single commercially available reagent, Fl-antidextran, a commercial kit for cell permeabilization, and the widely available FACS instrumentation to quantify bound antibody. Using an iron-based stain and microscopy, we previously concluded that 100 or 30 μg/mL Feridex and 3 μg/mL LFA produced iron-loaded cells with an internalized, cytoplasmic form of iron (see Figure 3 of Montet-Abou and colleagues¹²). However, the permeabilization shift assay indicates that the Feridex-LFA results in dextran nanoparticles adhering to the cell surface rather than being internalized (see Figure 2C). We conclude (1) that microscopy cannot be used to determine whether the cell-associated dextran nanoparticles of Feridex have been internalized or are adsorbed to the cell and (2) that LFA is not an acceptable transfection agent for loading cells with Feridex.

The ability of TFAs to increase cell-associated iron has been the subject of numerous publications employing numerous TFAs, cell types, and labeling conditions.^5,6,15–18 For our initial study on the stability of Feridex/TFA mixtures, we selected three of the most extensively described TFAs: poly-l-lysine, ProS, and LFA.¹² As shown here, LFA produces the adsorption of Feridex to the cell surface and hence should be rejected. The success of ProS at inducing Feridex internalization and the factors discussed below indicate that ProS is favored over poly-l-lysine for Feridex-based cell loading.

Figure 4.

Magnetic retention and permeabilization shift assays for different types of cells loaded with Feridex (Fe) and protamine sulfate (ProS). A, The percentages of iron-loaded (magnetically retained) cells and non–iron-loaded (not retained) cells are shown for H9c2, BT20, and HeLa cells. B, The shift in fluorescence with permeabilization is shown as the difference between the white bar (not permeabilized, NP) and the black bar (permeabilized, P).

Cytotoxicity of Poly-l-Lysine

Poly-l-lysine exhibits considerable cytotoxicity.¹⁹

Membrane-Translocating Activity of ProS

ProS relies on the guanidinium group of arginine for its positive charge, which, unlike the primary amino group of poly-l-lysine, is associated with the membrane-translocating activity of peptides.^20–22 The membrane-translocating activity of protamine is equal to or superior to that of the tat peptide; when both were conjugated to nanoparticles, the resulting protamine nanoparticle was internalized to higher levels than that of the tat nanoparticle.²¹

Size and Consistency of Material

ProS is a natural product obtained from salmon roe consisting of four peptides, all of which are about 4 kDa. Poly-l-lysine is prepared by polymerization to yield fractions with sizes varying from several kilodaltons to over 300 kDa.¹⁵ A poly-l-lysine greater than 300 kDa has been recommended.⁵ ProS is smaller than poly-l-lysine and is a highly consistent mixture.

Literature on the Clinical Use of Protamine

ProS has well-characterized toxicologic, pharmacologic, and biologic effects owing to its long-standing clinical uses of reversing the effects of heparin and as an excipient in insulin injections.

Table 2.

Summary of Transfection Agents Employed to Obtain Feridex-Loaded Cells

Transfecting Agent	Composition and Size	Stabilty of Feridex/TFA Mixture	Feridex Intern alization *	Cyto toxicity	Peptide MTS ^†
Protamine sulfate	Arginine, 4 kDa mixture	Only at low concentrations; see Montet-Abou et al¹² and Table 1	Internalized (see Figure 2)	Not reported	Yes; see Reynolds et al²¹
Poly-l-lysine	Lysine, variable	Only at low concentrations; see Montet-Abou et al¹²	Not reported	Yes; see Sgouras and Duncan¹⁹	No; see Wender et al²²
Lipofect amine	Positively charged lipid, 400–2,200 nm	Over wide concentration range; see Montet-Abou et al¹² and Table 1	Adsorbed (see Figure 2)	Not reported	Not reported

MTS = membrane-translocating sequence; TFA = transfection agent.

As shown by the permeabilization shift assay.

†

Similar to that of the tat peptide.

Sterility and Lack of Pyrogens

Mixtures of Feridex and ProS maintain the sterility and lack of pyrogens of the parent pharmaceuticals.

A summary of the three TFAs we have employed, their physical properties, and the results we obtained with them is provided in Table 2.

ProS can be employed to obtain iron-loaded cells, either by preparing Feridex/ProS mixtures as we and others have done^7,12,14 or by conjugation of ProS to crosslinked iron oxide (CLIO) magnetic nanoparticles, to obtain a material termed Pro-CLIO(Cy5.5).²¹ To date, a direct comparison of the two methods has been hindered by a lack of an optimized labeling method using ProS/Feridex mixtures, that is, one that avoids both TFA-induced nanoparticle precipitation and dextran adsorption to the cell surface. However, even without such a comparison being available, it is clear that each method has advantages over the other in some situations. Unlike ProS/Feridex mixtures, ProS-CLIO(Cy5.5) is a 30 to 50 nm nanoparticle that does not precipitate over a wide concentration range (1 and 500 mg/Fe mL). This is an advantage when loading suspended cells such as lymphocytes since the cells must be separated from the non–cell-associated iron by centrifugation and Feridex/TFA aggregates coprecipitate with cells. Unlike suspended cells, adherent cells can be washed by repeated media removal. A second advantage for the Pro-CLIO(Cy5.5) nanoparticle is its fluorescence, which enables nanoparticle distribution in a cell population to be determined by FACS and the fate of particles to be visualized by fluorescence microscopy. Whereas cell labeling with ProS-CLIO(Cy5.5) has advantages for loading suspended cells and in animal imaging studies, the Feridex/ProS method has an advantage for use in a clinical setting. ProS-CLIO(Cy5.5) is a research material that would require improvements in process design to attain the sterility and freedom from pyrogens present with mixtures of Feridex and ProS.

Given the different attributes of Feridex/TFA and Pro-CLIO(Cy5.5)-based cell labeling, the continued development of both Feridex/ProS mixtures and protamine-nanoparticle conjugates seems warranted. Use of this permeabilization shift assay will permit further optimization of cell labeling protocols designed to achieve both high levels of iron-loaded cells and a lack of surface-adsorbed nanoparticle with both Pro-CLIO(Cy5.5) and Feridex/ProS mixtures.

Footnotes

Acknowledgment

We are grateful to Dr. Evan Snyder for supplying the C17.2 cells.

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Cell Internalization of Magnetic Nanoparticles Using Transfection Agents

Abstract

Materials and Methods

Materials

Cell Culture

TFA-Assisted Feridex Loading

Feridex/TFA Stability Assay

Magnetic Cell Retention Assay

Permeabilization Shift Assay

Microscopy (Immunohistochemistry)

Microscopy (Fluorescent)

Results

Discussion

Cytotoxicity of Poly-l-Lysine

Membrane-Translocating Activity of ProS

Size and Consistency of Material

Literature on the Clinical Use of Protamine

Sterility and Lack of Pyrogens

Footnotes

Acknowledgment

References