Stability, accumulation and cytotoxicity of an albumin-cisplatin adduct

Abstract

The accumulation and cytotoxicity of a 10 μmol L⁻¹ equimolar human serum albumin-cisplatin adduct (HSA-Pt) was investigated in suspension Ehrlich Ascites Tumor Cells (EATC) and adherent Ehrlich Lettré Ascites Cells (Lettré). HSA-Pt did not induce apoptosis nor was it taken up by the cells to any significant amount within 24 h incubation. The accumulation and cytotoxicity of HSA-Pt was compared to 10 μmol L⁻¹ cisplatin for which a larger accumulation and cytotoxicity were observed in EATC compared to Lettré. The experiment was performed with cell medium exchange every fourth hour as HSA-Pt and cisplatin were not stable in RPMI-1640 with 10% serum. The stability was determined using size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and after 4 h new platinum peaks were observed. These findings indicate that before conducting cell experiments, the stability of the compound in the cell medium should be investigated especially when long exposure times are applied. Furthermore, HSA-Pt was found to be stable in Hanks Balanced Saline Solution (HBSS) and in Phosphate Buffered Saline (PBS) at pH 5.3, 6.1 and 7.4. Thus, the shift in pH when HSA-cisplatin passes from blood (pH 7.4) to tumor tissue (pH 5–6) is not capable of releasing cisplatin from HSA.

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Introduction

Platinum complexes are used successfully in the treatment of some solid tumors, but intrinsic or acquired resistance impairs the outcome of the treatment. The target of cisplatin is DNA in the nucleus. When cisplatin enters the cell the chloride ions are exchanged with water, as the chloride concentration inside the cell is lower than the extracellular concentration of 104 mmol L⁻¹.^1–4 Binding to DNA causes a bend of DNA which inhibits transcription and in the end leads to apoptosis. Treatment with cisplatin is often accompanied by severe side-effects as nephrotoxicity, ototoxicity and neurotoxicity, which may lead to termination of the treatment. Therefore, focus is on development of new platinum based drugs with improved efficacy and minimal side-effects.^5,6 Platinum is a soft metal and therefore, it has a high affinity towards soft ligands such as sulfur.⁷ Most platinum based drugs are administered intravenously and in the blood, they bind to proteins. The consequence of protein binding has given rise to debate. Protein binding may on one hand cause side-effects or a deactivation of the drug, as only the free drug is effectual. On the other hand, protein binding may act as a drug reservoir or lead to an enhanced accumulation in the tumor tissue through the Enhanced Permeability and Retention (EPR) effect. The EPR effect arises through an increased number of disorganized and leaky blood vessels in tumor tissue, which not only support the high energy requirement in the tumor tissue, but also enables large molecules to escape from the blood into the tumor tissue, where the pH is slightly acidic compared to the pH in the blood. Large molecules such as proteins will accumulate in the tissue as they are not drained by the lymphatic system.^8,9 Increased accumulation of radio-labeled albumin has been demonstrated in rat tumors.¹⁰ However, accumulation in tumor tissue does not necessarily result in cellular uptake as the compound has to pass the cell membrane. Proteins are taken up by the cells through different endocytosic pathways, which may be up or down regulated in different cell types.¹¹

Human Serum Albumin (HSA) is the most abundant serum protein with a serum concentration of 40–45 g L⁻¹ in healthy adults, corresponding to 0.6 mmol L⁻¹. HSA is a transport protein responsible for the transport of metals, fatty acids, thyroid hormones and drugs.¹² The nature of the binding between cisplatin and HSA/BSA has been described as negligible reversible,^13,14 irreversible,¹⁵ reversible¹⁶ or combinations thereof. The discrepancy between the description between cisplatin and HSA/BSA binding may be explained by differences in the protein to cisplatin ratios, incubation time, temperature and incubation medium as well as different analytical methods.

Exposure of, e.g., EATC suspensions cells to free cisplatin has recently been demonstrated to induce significant apoptosis within 18 h as evidenced by reduction in cell volume and activation of caspase-3 activity.¹⁷ Caspases are responsible for breakdown of cellular compounds related to DNA repair. The cytotoxicity of protein-bound Pt drugs has also been investigated. A phase 1 trial with terminal squamous cell carcinoma patients showed that cisplatin bound to HSA was not as efficient as conventional cisplatin treatment, although HSA-Pt had potential as palliative treatment as few side-effects were observed.¹⁸ In another study, comparing carboplatin and oxaliplatin to the corresponding HSA bound analogs, the cytotoxicity of HSA bound analogs decreased significantly in a cisplatin sensitive cell line, but showed comparable activity in a resistant cell line. The cellular accumulation diminished with a factor of 4–7, when the drug was bound to HSA (based on an estimate from the figure).¹⁹ Hoshino et al. showed that HSA-Pt and transferrin-cisplatin have antiproliferating properties, however, to a minor extent compared to free cisplatin.²⁰ Another paper using NHIK 3025 cells found that the cytotoxicity of cisplatin was lost by binding to serum protein, and that protein-bound platinum was not able to permeate the cells²¹

Cisplatin binds to proteins in cell media. The cytotoxicity of cisplatin was lost upon incubation of cisplatin in RPMI medium for 2 weeks or one month²² and for Pt(IV) compounds binding to RPMI-1640 supplemented with serum was observed, 66.3–97.8% of the total Pt had a molecular weight below 30 kDa after incubation for 24 h.²³ The binding of cisplatin to proteins can be changed by adding a strong nucleophile. Addition of acetylcysteine and sodium thiosulfate resulted in release of cisplatin bound to plasma proteins and it also prevented binding to proteins.²⁴ Another study demonstrated that cisplatin bound to plasma proteins was partly released in the presence of large excess of sodium N,N¹-diethyldithiocarbamate.²⁵

The aim of this study was to investigate the stability of HSA-Pt and cisplatin in different cell growth media and in PBS at different pH values and to compare the accumulation and cytotoxicity of HSA-Pt and non-conjugated cisplatin. We used adherent Ehrlich Lettré cells (Lettré) and non-adherent Ehrlich Ascites Tumor Cells (EATC) as cisplatin induced apoptose is well characterized in the latter.

Experimental

Chemical and reagents

Albumin from human serum 96–99%, cis-diammineplatinum(II) dichloride, Roswell Park Memorial Institute medium (RPMI)-1640, Fetal calf serum (FCS), trypsin (10×) penicillin and streptomycin (Sigma-Aldrich, St Louis, MO, U.S.), Sodium dihydrogen phosphate anhydrous, Suprapur®, sodium chloride, p.a., HNO₃ 65% pro analysis (subboiled twice) (Merck, Darmstadt, Germany), sodium hydroxide (1.0/0.1 M prepared from 35% NaOH), HCl 30% and ammonia solution 28%, (Prolabo, VWR International, Pennsylvania, USA), Ammonium acetate, puriss, p.a., Riedel-de Haën (Seelze, Germany), Platinum standard, 1000 μg mL⁻¹ in 10% HCl, SCP Science (Clark Graham, Canada), ClinChek® Controls, Serum control, lyophilized for trace elements, level 1, range 7.1–11 μg Pt L⁻¹ (Recipe, Munich, Germany), Phosphate Buffer Saline (PBS) adjusted to either pH 5, 6, or 7.4. Lysis buffer: 150 mmol L⁻¹, NaCl (Merck, Darmstadt, Germany), 20 mmol L⁻¹ HEPES , 1 mmol L⁻¹ EDTA (Sigma-Aldrich, St Louis, MO, U.S.), 0.5% Triton X-100, (Pharmacia Biotech AB, Uppsala, Sweden), 1 mmol L⁻¹ NaVO₃ and 1% protease inhibitor cocktail, Complete Mini, (Roche, Hvidovre, Denmark), Hanks Balanced Buffer Solution (HBSS) 10×, (Invitrogen, Taastrup, Denmark), microcentrifuge (175.MP (Ole Doch, Hvidovre, Denmark) and Eppendorf centrifuge 5804 (Eppendorf, Hamburg, Germany). Purified water (>18.2 MΩ cm) from a Milli-Q deionization unit was used throughout the experiments (Millipore, Bedford, MA, U.S.)

Formation of HSA-Pt adduct

Fresh solutions of HSA and cisplatin in PBS were prepared and equimolar amounts were incubated 24 h at 37 °C. Before dialysis a small subsample was taken and analyzed by SEC-ICP-MS.

Dialysis was performed to remove any remaining free cisplatin after formation of HSA-Pt. A Float-a-Lyzer®, molecular weight cut-off 10 000 Da, 5 mL, Spectrum Laboratories, Inc (Rancho Dominguez, CA) was used as described by the manufacturer. PBS was used as dialysis buffer and was exchanged three times during dialysis, which in total lasted 24 h.

SEC-ICP-MS

A BioSep-SEC-S2000 column 300 × 4.6 mm with a BioSep-SEC-S2000 guard column 30 × 4.6 mm, (Phenomenex, Torrance, California, U.S.) on a Agilent 1100 series HPLC system equipped with a variable UV detector set at 280 nm (Agilent Technologies, Santa Clara, CA, USA) was used to separate HSA and cisplatin. The mobile phase was 50 mmol L⁻¹ ammonium acetate adjusted to pH 7.4 and the flow was 350 μL min⁻¹. The eluate from the column was directed to the ICP-MS (Elan 6000, Perkin Elmer Sciex, Concord, ON, Canada). Either a Micromist Nebulizer (Glass Expansion Pocasset, MA, U.S.) or PEEK Mira Mist nebulizer, (Burgener Research, Mississauga, Ontario, Canada) was used with a cyclonic spray chamber, PC³ (Elemental Scientific Inc, Omaha, NE, U.S). Nebulizer gas, lens voltage and RF power were optimized daily using a 10 μg L⁻¹ Pt standard in mobile phase. The settings were; 100 ms dwell time, 5 sweeps reading⁻¹, 1 reading replicate⁻¹ and 1450 readings, given a total run time of 25 min. The Pt signals were monitored at m/z 195 and 196. The column recovery was measured by injecting the sample with and without column (flow injection analysis) and calculating the relative ratio between the Pt areas obtained.

Stability of cisplatin and HSA-Pt adduct in PBS and RPMI-1640 medium

The stability of HSA-Pt was tested by diluting HSA-Pt 10 and 100 times, respectively, in PBS at pH 7.4, incubating the sample 24 h at 37 °C, and analyzing the sample with SEC-ICP-MS. Furthermore, the stability of HSA-Pt was analyzed after incubation in PBS at pH 5 and pH 6 for 2 h at 37 °C. The stability of cisplatin and HSA-Pt, respectively, in RPMI-1640 medium with 10% fetal calf serum (FCS) and 1% penicillin and streptomycin was analyzed by incubating 10 μmol L⁻¹ of cisplatin or HSA-Pt, respectively, at 37 °C. The stability of HSA-Pt and cisplatin was also investigated in RPMI-1640 without FCS added. Samples were drawn at 0, 4 and 24 h and analyzed using SEC-ICP-MS. Furthermore, the stability of HSA-Pt in HBSS was investigated after incubation for 18 h at 37 °C.

Incubation and fractionation of cells to estimate cisplatin accumulation and caspase-3 activity

Ehrlich Ascites Tumor Cell (EATC), suspension cells were seeded in suspension cell culture flasks, and Ehrlich Lettré Ascites, (Lettré), adherent cells were seeded in Cellstar T75 Cell Culture Flasks, Adherent, (Tc treated). The cells were grown to 80–90% confluence in RPMI-1640 with 10% FCS and 1% penicillin/streptomycin at 37 °C, 5% CO₂ and 100% humidity. Approximately 1–2 × 10⁶ cells were used for the accumulation and caspase-3-activity experiments. The cell medium was removed and fresh medium containing no cisplatin or HSA-Pt (control), 0.14 μmol L⁻¹ cisplatin, 10 μmol L⁻¹ cisplatin or 10 μmol L⁻¹ HSA-Pt, respectively, was added. Samples were incubated at 37 °C and cell medium was exchanged every fourth hour for 24 h. An experiment with 10 μM cisplatin incubated 24 h without exchange of cell medium was performed for comparison. EATC suspension cell were transferred to Cellstar centrifuge tube (Greiner bio-one, Frickenhausen, Germany) and centrifuged 45 s at 700 g and the old medium were discarded. Cell medium from adherent cells was discarded. A freshly prepared cisplatin or HSA-Pt solution in cell medium was added. To evaluate adsorption, flasks with no cells, were incubated with cisplatin and HSA-Pt and treated as described above.

After incubation, the medium from adherent Lettré cells was discarded and the cells were washed with PBS. The cells were released from the flask with trypsin. The cells were transferred to a centrifuge tube, centrifuged 45 s at 700 g at room temperature and the trypsin was removed. The cells were washed three times with PBS by successive centrifugations and the PBS used for the last wash was collected for determination of Pt. The suspension cells were transferred to a centrifuge tube and centrifuged 45 s at 700 g, room temperature and cells were washed three times with 5mL PBS and the last wash was collected for determination of Pt. The EATC and Lettré cells were lysed with 2 mL ice-cold lysis buffer and the protein concentration was determined in a 10 μL subsample using the DC Protein Assay (Bio-Rad Laboratories, Copenhagen, Denmark). The lysate was transferred to a micro centrifuge tube and was centrifuged for 5 min at 600 g, 4 °C. The pellet constituted the nuclear fraction. The supernatant was transferred to a new microcentrifuge tube and was centrifuged 15 min at 5500 g, 4 °C to obtain the mitochondrial fraction. The supernatant, constituting the cytosol, was collected in a new tube. The nuclear and the mitochondrial pellets were washed three times with NaCl/KCl (30 mM/120 mM) and the last wash was collected for determination of Pt.

Determination of total Pt in cytosol, pellet and washing solution

Total Pt in the pellets, cytosol and washing solution was determined using ICP-MS applying the settings shown in Table 1. It was discovered that a longer uptake time, 120 s instead of 60 s, was required to obtain equilibrium condition for HSA-Pt compared to cisplatin presumably due to slight adsorption of HSA-Pt in the tubing. The pellets were dissolved by adding 100 μL 65% HNO₃ and the pellet was left to dissolve at least 24 h at room temperature and vortexed carefully several times (Heidolph Instruments GmbH & Co. KG, Schwabach, Germany). All samples were diluted with 0.1% HCl and 0.65% HNO₃ to a concentration within the calibration curve, 0.1–20 μg Pt L⁻¹. Accuracy of the method was investigated by analyzing a serum control sample according to the manufacturer's protocol.

Table 1 ICP-MS settings for determination of total Pt

ICP-MS	Elan 6000
Nebulizer	Micromist (micro-concentric quartz nebulizer)
Nebulizer gas flow	1.04 L min^−1
RF power	1300 W
Lens voltage	9.8 V
Plasma gas flow	15 L min^−1
Auxiliary gas flow	1.2 L min^−1
Skimmer	Nickel
m/z monitored	192, 195, 196
Dwell time	100 ms
Number of channels/AMU	1
Sweeps/reading	25
Reading/replicate	1
Replicate	5

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Caspase-3 activity

Following treatment with cisplatin or HSA-cisplatin, cells were centrifuged at 1000 g for 6 min at 4 °C. The pellet was washed once with PBS, lysed in ice-cold lysis buffer, resuspended, and stored at −80 °C overnight. The cell lysates were subjected to three freeze-and-thaw cycles and 2 × 10 s sonication. The cell lysate was centrifuged at 20 000 g for 5 min and the supernatant transferred to new eppendorf tubes. Protein content in supernatants was determined (DC Protein Assay, Bio-Rad) and concentration was adjusted for activity measurements (4 μg μl⁻¹). Caspase-3 activity in cell lysates was estimated in 96-well plates using the ApoTargetCaspase 3/CPP32 Colorimetric assay (Invitrogen, Taastrup, Denmark) according to the manufacturer's protocol. Absorbance was measured at 405 nm using a microplate reader (Bmg LabTechnologies, Offenburg, Germany). Experiments were performed in triplicate.

Results and discussion

Stability HSA-Pt and cisplatin RPMI-1640 medium and PBS

The stability of cisplatin and HSA-Pt in RPMI-1640 medium and PBS was investigated by SEC-ICP-MS. Fig. 1 shows chromatograms of HSA-Pt after incubation and dialysis for 24 h as well as the chromatograms of a fresh cisplatin solution in PBS and cisplatin stored for 24 h in PBS. A complete separation of HSA-Pt (t_r 5.7 to 7 min) and cisplatin (t_r 12.5 min) was obtained. The chromatogram of HSA-Pt consists of three unresolved peaks presumably the monomer, dimer and trimer of HSA. These peaks are also observed in a SEC-UV chromatogram of HSA without the presence of cisplatin which can be seen in the bottom of Fig. 1. The chromatogram of cisplatin dissolved in PBS consists of several peaks. The most intense peak corresponds to cisplatin, whereas minor peaks are degradation products of cisplatin, resulting from exchange of chloride ions with water. Several degradation products can be formed depending on pH. When cisplatin was stored in PBS for 24 h, one new compound corresponding to 1.9% of the total Pt area was formed with a retention time of 10.2 min. Besides, only minor changes in the area distribution of the different peaks were observed, indicating that cisplatin is quite stable in PBS.

Fig. 1 SEC-ICP-MS chromatograms. (A) Cisplatin stored 24 h in PBS, the new formed compound is marked with an asterisk. (B) Cisplatin freshly dissolved in PBS. (C) HSA and cisplatin incubated 24 h at 37 °C and dialyzed for 24 h. (D) HSA in PBS analyzed with SEC-UV.

The column recovery for HSA-Pt was 108 ± 3.0% (n = 3) and the column recovery for cisplatin was 98.1 ± 0.75% (n = 3). A high and stable column recovery of both cisplatin and HSA-Pt is important as the amount of free cisplatin later is calculated relative to the total Pt area.

The stability of HSA-Pt in PBS, pH 7.4, was determined with SEC-ICP-MS after dilution 10 and 100 times, respectively and found to be stable for at least 24 h, as neither free cisplatin nor cisplatin degradation products were observed. Furthermore, the stability was examined after dilution with PBS, pH 5 and pH 6. No free cisplatin or degradation products were observed in the chromatograms after incubation for 2 h at 37 °C and it was concluded that HSA-Pt is also stable under slightly acidic conditions. Therefore, the pH change occurring in the passage from blood to tumor tissue will not release cisplatin from HSA. However, other factors in the tumor tissue might release cisplatin from HSA.

The stability of the test compound in the cell medium is important when studying cytotoxicity and cellular accumulation. The RPMI-1640 cell medium contains a variety of vitamins, amino acids and inorganic salts. FCS is often added to the cell medium as a nutrient source. Some of the compounds in the cell medium may interact with the test compound and alter it. The stability of cisplatin in RPMI-1640 with and without 10% FCS was tested and the chromatograms are shown in Fig. 2. After incubation of cisplatin in RPMI-1640 without serum, cisplatin was transformed to several new Pt containing compounds with retention times between those of HSA-Pt and cisplatin. When incubating cisplatin in RMPI-1640 with 10% FCS, cisplatin bound to the serum proteins and furthermore, the same compounds were formed as observed in RPMI-1640 without FCS. After incubation of cisplatin in RPMI-1640 medium for 24 h with and without 10% FCS, the amount of free cisplatin decreased from 95% to 25% and 36%, respectively. Already after incubation for 4 h, the percentage of cisplatin had declined to 77% and 82% with and without FCS, respectively. These results demonstrated that the stability of the compound in cell medium is very important especially when conducting cell experiments with long exposure time. Accumulation and cytotoxicity may be altered significantly or may be caused by another compound than the test compound. Furthermore, elucidation of the transport mechanism will be difficult, if the structure of the compound is unknown or altered during the experiment. The rate constant for the disappearance of cisplatin from the medium with and without FCS was estimated to 0.06 h⁻¹ and 0.04 h⁻¹, respectively, assuming first order kinetics, however only based on three time points. The disappearance of cisplatin in the cell medium has previously been determined using [¹H ¹⁵N] HSQC NMR, the rate constant for the hydrolysis was calculated to 0.205 h^−1 26 and rate constant for the disappearance of cisplatin and mono-aqua-cisplatin (both the protonated or deprotonated version) from the medium was calculated to 0.101 h⁻¹, which is faster than in our experiment. HSA-Pt was also unstable in RPMI-1640 medium both with and without 10% FCS added. The chromatogram in Fig. 3 shows the formation of small compounds with retentions times between those of HSA-Pt and cisplatin. The retention times correspond to those observed after incubation of cisplatin in RPMI-1640. The species have not been identified, but they could be reaction products between cisplatin and sulfur-containing compounds such as cystine and methionine from the cell medium. A carbonate–cisplatin adducts could also be present as this adduct has been identified using NMR. The carbonate adduct is formed due to the presence of dissolved CO₂ in the cell medium.²⁷

Fig. 2 SEC-ICP-MS chromatograms. A 10 μmol Pt L⁻¹ cisplatin incubated at 37 °C in RPMI-1640. B 10 μmol Pt L⁻¹ cisplatin incubated at 37 °C in RPMI-1640 with 10% FCS. t0, t4, and t24 indicate exposure time. The chromatograms are offset, same scale.

Fig. 3 SEC-ICP-MS chromatograms. (A) HSA-Pt in RPMI-1640 medium +10% FCS, (B) HSA-Pt in HBSS incubated 18 h at 37 °C. The new formed Pt-compounds are marked with asterisks.

After incubation of HSA-Pt in RPMI-1640 with FCS for 4 h, 97.2% of the total Pt area was detected as HSA-Pt, but after 24 h only 87.7% was detected as HSA-Pt. Therefore, it is not possible to conduct a 24 h accumulation experiment with HSA-Pt as a Pt containing compound is released from HSA during the experiment. It would not be possible to decide whether an observed accumulation or cytotoxicity was caused by HSA-Pt or its degradation products. Therefore, the cell medium was exchanged every fourth hour. In this way, after 4 h HSA-Pt degradation products would at most comprise of 2.8% of the total Pt, corresponding to 0.28 μmol Pt L⁻¹, and during the experiment, an average concentration of HSA-Pt degradation products would be around 0.14 μmol Pt L⁻¹. Control experiment with 0.14 μmol Pt L⁻¹ cisplatin concentration was conducted to estimate the cytotoxicity and accumulation of a low concentration of cisplatin.

In contrast, HSA-Pt was stable after incubation for 18 h at 37 °C in HBSS cell medium and in NH₄HCO₃, as the chromatogram in Fig. 3 only shows HSA-Pt. HBSS contains, in contrary to RPMI-1640, no vitamins or amino acids, but only salts and this could explain the observed stability in this cell medium.

Cellular accumulation and cytotoxicity of HSA-Pt and cisplatin

The Pt accumulation in EATC and Lettré cells was measured using ICP-MS. Three Pt isotopes were measured and the amount of Pt was calculated based on the average concentration of each isotope. Excellent agreement between the three isotopes was found with RSD < 1%. Table 2 shows the sensitivity, precision, instrumental LOD, LOQ, repeatability and accuracy based on ¹⁹⁵Pt. The analytical figures of merit show that the ICP-MS method is linear and provide precise and accurate results. As no cell reference material with Pt was available, the accuracy of the method was tested by analyzing a serum reference sample.

Table 2 Analytical figures of merit for the determination of total Pt based on ¹⁹⁵Pt

Linearity (0.1–20 μg Pt L^−1) R^2	0.99999
Sensitivity 1 μg L^−1 (^195Pt)	13 500 cps
Instrumental Limit of Detection calculated as YB + 3·sb	25 ng L^−1 (n = 3)
Instrumental Limit of Quantification calculated as YB + 10·sb	60 ng L^−1 (n = 3)
Repeatability over 4 h (Pt standard 1 μg Pt L^−1)	0.53% (n = 4)
Accuracy (serum control) range 7.1–11 μg L^−1 Pt	10.2 ± 0.7 μg L^−1 (n = 3)

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Table 3 shows the amount of Pt in pg found in the pellet and cytosol, the protein contents as a measure of the cell number and the caspase-3-activity as a measure of cytotoxicity. It has previously been demonstrated that the nucleus is completely separate from the cytosol using this cell fractioning method.²⁸ All measurement is based on one experiment and therefore no standard deriviation can be calculated. Based on previously accumulation experiments with 10 μmol L⁻¹ cisplatin incubated 18 h, the RSD is around 25% on four independent experiments. The amount of Pt found in the mitochondrial fraction was low. Due to the risk of the mitochondrial fraction being contaminated with slight amounts of nucleus fraction, the amount of Pt determined in the nucleus and mitochondrial fractions were reported as the sum, termed the pellet, though they were measured separately. The amount of Pt found in the pellet and cytosol after treatment with HSA-Pt was only slightly higher than the control and not higher than the amount found after treatment with 0.14 μmol L⁻¹ cisplatin. This means that cisplatin bound to HSA is not taken up by EATC or Lettré cells. According to a microarray analysis both cells lines express the membrane associated molecules required to perform endocytosis, e.g. clathrin and caveolae (unpublished data), hence they should be able to take up proteins by endocytosis. The lack of uptake of HSA-Pt could be explained by conformational changes of HSA upon binding to cisplatin, which could hinder endocytotic uptake of HSA-Pt. It has previously been shown that a HSA-cisplatin adduct (1 : 1) incubated 24 h, formed dimer and higher polymer forms of HSA and it was suggested that the polymerization influences biological activity.²⁹ We supposed that the HSA-Pt adduct used in this experiment is similar to the above mentioned, as similar incubation ratio and time was used, however there was a slight difference in the pH of the incubation buffer, 6.4 versus 7.4. It has previously been shown that the cytotoxicity of cisplatin encapsulated in nanoparticles was strongly dependent on the cell line,³⁰ Thus, the uptake of cisplatin is cell line, concentration and time dependent.

Table 3 Cellular accumulation and cytotoxicity of HSA-Pt and cisplatin in EACT and Lettré

	Pellet pg Pt	Cytosol pg Pt	Protein μg	Pellet pg Pt μg protein^−1	Cytosol pg Pt μg protein^−1	Caspase-3-activity relative to control
EATC (suspension cells)
Control	84	2020	2148	4 × 10^−2	0.9	1.0
10 μmol L^−1 HSA-cisplatin	246	4226	2509	0.10	1.7	1.0
10 μmol L^−1 cisplatin	59 687	207 838	1133	50.9	183	5.0
0.14 μmol L^−1 cisplatin	516	4038	1935	0.23	2.1	0.5
Lettré (adherent cells)
Control	24	1744	810	3 × 10^−2	2.1	1.0
10 μmol L^−1 HSA-cisplatin	317	2665	1527	0.2	1.8	0.9
10 μmol L^−1 cisplatin	18 233	138 107	1159	15.7	119	1.4
0.14 μmol L^−1 cisplatin	355	7484	1147	0.3	6.5	0.6

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The amount of cisplatin found in the cytosol was 183 and 119 pg Pt μg protein in EATC and Lettré, respectively. In comparison with other experiments, our results show a larger accumulation presumably due to a longer exposure time. In lysates from T289 malignant melanoma cells (adherent cells) exposed to 50 μg ml⁻¹ cisplatin (160 μmol L⁻¹) for 1 h, 52–109 pg Pt μg⁻¹ protein was found.³¹ In human mammary adenocarcinoma cells (MCF-7), the cellular accumulation of cisplatin was 10 pg Pt μg⁻¹ after 2 h exposure of 10 μmol L⁻¹ cisplatin at 37° C (estimated from figure).³² The average cellular concentration of Pt in SW480 cells (adherent cells) after exposure to 3 μmol L⁻¹ cisplatin for 2 h was 5 pg Pt μg⁻¹ protein.³³

The cellular accumulation of 10 μmol L⁻¹ cisplatin in the two cell lines is quite different, especially the amount of Pt found in the pellet from EATC is notably higher than the amount found in Lettré cells. The higher accumulation in EATC also correlates with an increased caspase-3-activity. In concordance with previous experiments, 10 μmol L⁻¹ cisplatin only induce minor apoptosis in Lettré cells.³⁴ This may be caused by a different spatial localization of membrane transporters in Lettré as adherent cells are polarized consisting of a basolateral and an apical domain.³⁵ The low cisplatin concentration 0.14 μmol L⁻¹ does not induce apoptosis.

When measuring the cellular accumulation of Pt, care must be taken not to overestimate it. Therefore, the nucleus and the mitochondria were washed three times with PBS and the amount of Pt in the third washing solution was below the LOD. This demonstrates that the amount of Pt found in the fractions originates from the inside and that three washing step are sufficient to remove Pt from the outside. There is, however, a minor risk of losing Pt during the washing procedure as the organelles are not completely tight. It has been shown by Egger et al. that adsorption of the test compound to the wells can lead to overestimation of the cellular accumulation due to desorption of the test compound, when the cell are lysed directly in the wells.³³ In our experiments, adherent cells were released from the flasks using trypsin and the medium was discarded. The cells were washed three times with PBS before lysis to remove any test compound desorbed from the flasks. Furthermore, adsorption of HSA-Pt and cisplatin to the flasks was investigated and found to be negligible (data not shown).

The amount of protein is a measure of the cell number. The amount of protein found in EATC treated with 10 μmol L⁻¹ cisplatin was only half of that found in control samples and in samples with the low cisplatin concentration or HSA-Pt. This corresponds well with the caspase-3-activity being significantly higher, indicating that a fraction of the cells have died. The amount of protein found in EACT and Lettré after treatment with 10 μmol L⁻¹ cisplatin without medium exchange was 117% and 122% of that found after medium exchange every 4 h. This means that some cells probably are lost during medium change or that more cells have died when the medium was exchanged frequently. When cells die, they are detached from the surface and may be lost, when the medium is exchanged. However, the amount of Pt as well as caspase-3-actively is calculated relative to the amount of protein and thereby it is corrected for.

Conclusion

HSA-Pt was not taken up by EATC or Lettré cells, nor did it induce cytotoxicity. Cisplatin was taken up by both cell lines, however, to a notably larger extent in EATC, where it also showed greater cytotoxicity. HSA-Pt was found unstable in RPMI-1640 with and without 10% FCS, but, HSA-Pt was stable in PBS at pH 5.3, 6.1 and 7.4 and in HBSS. Cisplatin was unstable in RPMI-1640 with and without 10% serum. The experiments show that the stability of the test compound in the cell medium should be investigated before performing cell experiments.. The test compound might interact with constituents in the cell medium and therefore test solutions should be changed repetitively, especially if long exposure time is used.

List of abbreviations

BSA	Bovine Serum Slbumin
EATC	Ehrlich Ascites Tumor Cells
EPR	Enhanced Permeability and Retention
FCS	Fetal Calf Serum
HBSS	Hanks Balanced Saline Solution
HSA	Human Serum Slbumin
HSA-Pt	Albumin-cisplatin adduct
HSQC NMR	Heteronuclear Single Quantum Coherence Nuclear Magnetic Resonance
Lettré	Adherent Ehrlich Lettré Ascites Cells
LOD	Limit of Detection
LOQ	Limit of Quantification
PBS	Phosphate Buffered Saline
RPMI-1640	Roswell Park Memorial Institute medium-1640
SEC-ICP-MS	Size Exclusion Chromatography-Inductively Coupled Plasma-Mass Spectrometry

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