Exosomes loaded with ultrasmall Pt nanoparticles: a novel low-toxicity alternative to cisplatin | Journal of Nanobiotechnology



Cell culture

Human placental mesenchymal stem cells (hpMSCs) were purchased to Cellular engineering Technologies (CET (Coraville, IA, USA)). Metastatic murine skin melanoma cells (B16-F10 cells) and glioblastoma cells (U251-MG) were provided by cell services from Cancer Research-UK. They were grown in Dulbecco’s modified Eagle’s medium (DMEM, Biowest, France) supplemented with 10% fetal bovine serum (FBS, GIBCO, USA), 1% penicillin/streptomycin and 1% amphotericin (Biowest, France). For culturing hpMSCs, 5 µg mL−1 of FGF-2 growth factor (PeproTech, USA) were also added to the cell culture media. hpMSCs were maintained under hypoxic conditions whereas U251-MG cells and B16-F10 cells were cultured under and normoxic conditions.

To obtain the culture media free of exosomes, they were depleted from FBS by ultracentrifugation (100,000g, 8 h, 4 °C).

Exosome purification

For exosome purification, cell lines were seeded in cell culture plates until they reached the 80% of confluence. After that, the cell culture medium was replaced by medium free of exosomes and cells were maintained for another 48 h. Then, ExosB16−F10, ExoshpMSCs and ExosU251−MG were isolated by serial centrifugation cycles from cell culture supernatants of source cells. In order to remove cell remaining debris, supernatants were centrifuged at 2000g during 20 min at 4 °C. For the separation of the microvesicles, samples were then centrifuged during 1 h at 10,000g at 4 °C. To finally isolate the exosomes, samples were ultracentrifuged twice for 2 h at 100,000g at 4 °C. The final pellets were dispensed in PBS for the following analysis.

Synthesis of Pt-Exo

The purified exosome fraction resuspended in PBS was treated with a solution of H2PtCl6 (Mw 409.81 g/mol) at room temperature for 12 h to maximize the diffusion and incorporation of the Pt4+ ions in the exosomal lumen. To modulate the amount of PtNPs generated inside the exosomes, several precursor concentrations were employed (0.06; 0.12; 0.3 and 0.6 mM of H2PtCl6). The mixtures were then ultracentrifuged in order to discard the non-internalized Pt species (100,000g, 2 h, 4 °C). Pt-ExosB16−F10, Pt-ExoshpMSCs and Pt-ExosU251−MG were finally resuspended in PBS and treated in a 6 bar CO atmosphere during 40 min using a high-pressure Teflon-lined autoclave under gently stirring at 30 °C [48]. After the treatment, and before opening the autoclave, CO was desorbed with air to keep the resulting Pt-Exos in a neat environment.

Characterization of Exos and Pt-Exos

Both biological and physicochemical techniques were used to characterized loaded and empty exosomes. A Pierce BCA protein assay (Thermo Fisher Scientific, USA) was performed in order to quantify the protein content in the exosomal sample according to manufacturer instructions. Western Blot was carried out in order to analyze the exosomal protein content. To do that, 10 µg of exosomes were lysed and precipitated using RIPA buffer and cold acetone (1:1 v/v) at − 20 °C for 2 h. Then, 5 µL of Laemmly buffer were added to the sample and boiled during 5 min at 95 °C. Proteins were subsequently separated using a 12% SDS-PAGE at 100 V for 2 h at room temperature and transferred to a nitrocellulose membrane at 4 °C for 4 h (300 mA). Blots were then blocked overnight at 4 ºC with 5% milk-TBS buffer. Then, they were incubated with CD9 (1:2000, Abcam) and CD91 (1:500, Santa Cruz Biotechnology) antibodies for 1.5 h. The membranes were finally washed three times with TBS Tween and incubated with the secondary antibody (anti-HRP, Sigma Aldrich) before being imaged by chemiluminescence. NTA analysis (Nanosight NS200, Malvern Panalytical) was used to determine the diameter and the concentration (expressed in particles/mL) of Exos and Pt-Exos. TEM (T20-FEI Technai transmission electron microscopy) imaging was also employed for evaluate the morphology, shape and size of the loaded and empty exosomes. The microscope was operated at 200 kV with a LaB6 electron source fitted with a SuperTwin objective lens allowing a point-to-point resolution of 2.4 A. A solution of 3% phosphotungstic acid was employed as contrast agent to visualize the exosomes. An Analytical Titan (FEI company) high resolution transmission electron microscope with a spherical aberration corrector was used for HAADF-STEM imaging at 300 kV. An elemental analysis of the Pt-Exos was carried out by EDX. Zeta potential of Pt-ExoshpMSCs and Pt-ExosU251−MG was measured by dynamic light scattering (DLS) at pH = 7 in a Brookhaven 90 Plus equipment using the ZetaPals software. The amount of Pt inside the vesicles was measured using microwave plasma atomic emission spectrometry (4100 MP-AES, Agilent Technologies, USA) and normalized by the total protein amount of Pt-Exos. To do that, samples were digested with 10% aqua regia (HNO3 + 3HCl) in 1.5 mL of dH2O during 2 h at room temperature. Calibrations were carried out employing a Pt standard in 10% aqua regia ranging from 0 to 10 ppm.

Study of Pt-Exos in vitro cytotoxicity

The therapeutic properties of Pt-Exos as antiproliferative and cytotoxic agents in different cell cultures were first determined by using the Blue Cell Viability assay (Promega, United States) as previously reported. The cytotoxicity produced by 4; 2; 1; 0.5; 0.25 and 0.125 µg of Pt-ExoshpMSCs/100 µL was evaluated when incubating them following three different conditions. Approach 1: cells were incubated only with the Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein). Approach 2: cells were treated with Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein) containing NH4Cl, and approach 3: cells were treated with the Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein) with NH3 within them.

In a different experiment, 4; 2; 1; 0.5; 0.25; 0.125 µg of exosomes/100 µL loaded with different amount of PtNPs (0.560 and 0.86 µg of Pt/µg of exosomal protein) were incubated with the cells during 48 h. Exosomes loaded with 0.560 and 0.86 µg of Pt/µg of exosomal protein are named as 5x-Pt-ExoshpMSCs and 10x-Pt-ExoshpMSCs, respectively. The results are expressed as cell viability percentage, considering as 100% the non-treated cells. Cell cultures incubated with the highest dose of exosomes were also observed by inverted conventional microscopy (Olympus IX81) in order to evaluate their morphology. All the experiments were performed in triplicate.

In vitro cell death evaluation

To study in more detail the efficiency of Pt-Exos as an antiproliferative and a cytotoxic tool, hpMSCs were seeded onto a 96-well plate (5000 cells/well). After 24 h in culture, 4 and 2 µg of exosomes/100 µL were added (both from 5x-Pt-ExoshpMSCs and 10x-Pt-ExoshpMSCs). Then, after 48 h, cell culture media was discarded and cells were washed twice with PBS. The cytotoxicity produced by the Pt-Exos loaded with different amounts of PtNPs was determined with the LIVE/DEAD kit (Thermo Fisher Scientific, USA) using an inverted fluorescence microscope (Olympus IX81) and following manufacturer instructions.

The selectivity and the efficiency of Pt-Exos as therapeutic vectors were quantified by flow cytometry (FACSAria BD cytometer, BD Bioscience). To determine the effect of the Pt-Exos on the proliferation rate of the different cell types, the distribution of the cell-cycle phases after the incubation of the exosomes was assessed. First, cells were seeded onto 6-well plates at a density of 250,000 cells/well. After 24 h, 2 µg of 10x-Pt-ExoshpMSCs and 10x-Pt-ExosU251−MG were incubated with both hpMSCs and U251-MG cells during 48 h. Then, cells were trypsinized and washed twice with PBS (1500g, 5 min). The final cell pellet was suspended in 200 µL of PBS and was fixed with 70% ice-cold ethanol and maintained at 4 °C in these solution for 24 h more before being analyzed under the flow cytometer. DNA staining was carried out by adding RNase A and propidium iodide to the cell solution. Control samples (non-treated cells and cells treated with the exosomes but in the absence of PtNPs within them) were also run to know the standard distribution of cells cycles in the cell lines assayed.

The analysis of cell apoptosis and necrosis was developed through a double-staining with Annexin V-FITC (5 µL) and propidium iodide (5 µL) during 15 min prior to their analysis by flow cytometry (FACSARIA BD equipment and FACSDIVA BD software).

In vivo experiments

The procedures performed in this study were previously approved by the Project License PI07/21 and PI43/21 by the Ethic Committee for Animal Experiments from the University of Zaragoza (Comisión Ética Asesora para la Experimentación Animal de la Universidad de Zaragoza). Mice were fed ad libitum and their maintenance and care under specific pathogen-free conditions were performed accordingly with the Spanish Policy for Animal Protection RD53/2013 and the European Union Directive 2010/63 regarding the protection of animals destined to experimental and other scientific purposes.

In this study, six-to eight-week old female BALB/c nu/nu mice (Envigo) were used. All the animals were maintained seven days under quarantine as soon as they arrived to the animal facilities and before starting the experiments. For the induction of the xenograft tumor, animals received a subcutaneous injection of 7 × 106 U251-MG cells in 200 µL of saline. To evaluate the potential weight loss or pain symptoms, animals were weighed and monitored daily. Tumor sizes were measured with a caliper every two days. The manipulation of the animals was always performed under sterile conditions in a hood.

After 11 days of tumor implantation, 100 µg of Pt-Exos were both, IV (in the tail vein) and IT, administered. Alternatively, cisplatin (a clinical approved drug) was administered as positive control to compare the effectiveness of the Pt-Exos based therapy. The animal groups included in the study are shown in Additional file 1: Table S1.

In order to compare the therapeutic effect produced by cisplatin and the Pt-Exos, the administered dose was calculated in order to inject the same amount of Pt:

$$0.86\,\text{ug}\frac{Pt}{ug\,exosome}\cdot 100\,\text{ug}\,exosomes\,administered=86\,\text{ug}\,of\,Pt\,administered$$

Taking into account the Pt present in the cisplatin molecule:

$$86\,\text{ug}\,of Pt \cdot \frac{300.01\,\text{ug}\,molecule}{78\,\text{ug}\,Pt}=330\,\text{ug} \, of \, cisplatin\, administered$$

In the therapeutic study, once the control animals archive the maximum tumor size approved in the Ethical Committee, the experiment was finished and the animals were sacrificed. Euthanasia of animals was performed by CO2 inhalation. Selected organs were collected from each animal for histopathological analysis and to determine the amount of Pt delivered as a consequence of treatment. For histopathological analysis the samples were fixed in 4% paraformaldehyde (Alfa Aesar) for 24 h, followed by cold 70% ethanol. The experimental plan is included in Fig. 1.

Fig. 1
figure 1

Experimental procedure followed in the in vivo experiments. Tumor implantation, both administration routes of the two therapeutic complexes (Pt-ExosU251−MG and cisplatin), sacrifice of the animals and ex vivo analysis of the necropsies

Tissue samples were then embedded in paraffin and three-micrometer sections were stained with H-E. Immunohistochemical analysis was carried out by employing human Ki-67 antibody (Agilent) with the automated immunostaining platform Autostainer Link (Dako). Sections (4–5 um) were dewaxed and rehydrated with dH2O and antigen retrieval was performed by heat at pH = 7 during 20 min. Tumor samples were incubated with primary Ki-67 antihuman antibody for 20 min followed by the visualization system conjugated with horseradish peroxidase (Flex/HRP system Agilent). Hematoxylin staining was used as contrast. Finally, sections were dehydrated and permanently mounted for their visualization under a conventional inverted microscope (Olympus IX81). To obtain the percentage of positive cells, Ki-67 expression was assessed in all mice groups by counting positive and negative cells in three acquisition images from all animals from each group using ImageJ software.

For the biodistribution analysis, after 11 days of tumor implantation cisplatin or Pt-ExosU251−MG were injected in the tail vein and animals were sacrificed after 24 and 96 h injection. Both groups received a total amount of 86 µg Pt. In the control group tumors were induced however did not receive any Pt treatment. Pancreas, spleen, kidneys, liver, lungs, brain and tumor were collected, weighted and digested in a solution of aqua Regia during 7 days at room temperature. To evaluate platinum accumulation; the solutions were diluted (1/100) in miliQ H2O and total amount of the metal within the tissues was determined by ICP-MS (Perkin Elmer Elan DRC-e) in the Chemical Analysis Service from the University of Zaragoza.

Statistical analysis

All the results are expressed as mean ± SD. Statistical analysis of the data and the significant differences among the means were analyzed by two-way analysis of variance (ANOVA) for multiple comparisons by Dunnett’s multiple comparisons test (GraphPad Software). Statistically significant differences were expressed as follows: *p < 0.05; **p < 0.01; ***p < 0.0001 and ****p < 0.00001.