Technology

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

Human placental mesenchymal stem cells (hpMSCs) have been bought to Mobile engineering Applied sciences (CET (Coraville, IA, USA)). Metastatic murine pores and skin melanoma cells (B16-F10 cells) and glioblastoma cells (U251-MG) have been offered by cell companies from Most cancers Analysis-UK. They have been 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 development issue (PeproTech, USA) have been additionally added to the cell tradition media. hpMSCs have been maintained underneath hypoxic situations whereas U251-MG cells and B16-F10 cells have been cultured underneath and normoxic situations.

To acquire the tradition media freed from exosomes, they have been depleted from FBS by ultracentrifugation (100,000g, 8 h, 4 °C).

### Exosome purification

For exosome purification, cell strains have been seeded in cell tradition plates till they reached the 80% of confluence. After that, the cell tradition medium was changed by medium freed from exosomes and cells have been maintained for one more 48 h. Then, ExosB16−F10, ExoshpMSCs and ExosU251−MG have been remoted by serial centrifugation cycles from cell tradition supernatants of supply cells. With the intention to take away cell remaining particles, supernatants have been centrifuged at 2000g throughout 20 min at 4 °C. For the separation of the microvesicles, samples have been then centrifuged throughout 1 h at 10,000g at 4 °C. To lastly isolate the exosomes, samples have been ultracentrifuged twice for two h at 100,000g at 4 °C. The ultimate pellets have been disbursed in PBS for the next evaluation.

### Synthesis of Pt-Exo

The purified exosome fraction resuspended in PBS was handled with an answer of H2PtCl6 (Mw 409.81 g/mol) at room temperature for 12 h to maximise the diffusion and incorporation of the Pt4+ ions within the exosomal lumen. To modulate the quantity of PtNPs generated contained in the exosomes, a number of precursor concentrations have been employed (0.06; 0.12; 0.3 and 0.6 mM of H2PtCl6). The mixtures have been then ultracentrifuged with a purpose to discard the non-internalized Pt species (100,000g, 2 h, 4 °C). Pt-ExosB16−F10, Pt-ExoshpMSCs and Pt-ExosU251−MG have been lastly resuspended in PBS and handled in a 6 bar CO environment throughout 40 min utilizing a high-pressure Teflon-lined autoclave underneath gently stirring at 30 °C [48]. After the remedy, and earlier than opening the autoclave, CO was desorbed with air to maintain the ensuing Pt-Exos in a neat setting.

### Characterization of Exos and Pt-Exos

Each organic and physicochemical methods have been used to characterised loaded and empty exosomes. A Pierce BCA protein assay (Thermo Fisher Scientific, USA) was carried out with a purpose to quantify the protein content material within the exosomal pattern in line with producer directions. Western Blot was carried out with a purpose to analyze the exosomal protein content material. To try this, 10 µg of exosomes have been lysed and precipitated utilizing RIPA buffer and chilly acetone (1:1 v/v) at − 20 °C for two h. Then, 5 µL of Laemmly buffer have been added to the pattern and boiled throughout 5 min at 95 °C. Proteins have been subsequently separated utilizing a 12% SDS-PAGE at 100 V for two h at room temperature and transferred to a nitrocellulose membrane at 4 °C for 4 h (300 mA). Blots have been then blocked in a single day at 4 ºC with 5% milk-TBS buffer. Then, they have been incubated with CD9 (1:2000, Abcam) and CD91 (1:500, Santa Cruz Biotechnology) antibodies for 1.5 h. The membranes have been lastly washed 3 times with TBS Tween and incubated with the secondary antibody (anti-HRP, Sigma Aldrich) earlier than being imaged by chemiluminescence. NTA evaluation (Nanosight NS200, Malvern Panalytical) was used to find out the diameter and the focus (expressed in particles/mL) of Exos and Pt-Exos. TEM (T20-FEI Technai transmission electron microscopy) imaging was additionally employed for consider the morphology, form and dimension of the loaded and empty exosomes. The microscope was operated at 200 kV with a LaB6 electron supply fitted with a SuperTwin goal lens permitting a point-to-point decision of two.4 A. An answer of three% phosphotungstic acid was employed as distinction agent to visualise the exosomes. An Analytical Titan (FEI firm) excessive decision transmission electron microscope with a spherical aberration corrector was used for HAADF-STEM imaging at 300 kV. An elemental evaluation of the Pt-Exos was carried out by EDX. Zeta potential of Pt-ExoshpMSCs and Pt-ExosU251−MG was measured by dynamic gentle scattering (DLS) at pH = 7 in a Brookhaven 90 Plus gear utilizing the ZetaPals software program. The quantity of Pt contained in the vesicles was measured utilizing microwave plasma atomic emission spectrometry (4100 MP-AES, Agilent Applied sciences, USA) and normalized by the overall protein quantity of Pt-Exos. To try this, samples have been digested with 10% aqua regia (HNO3 + 3HCl) in 1.5 mL of dH2O throughout 2 h at room temperature. Calibrations have been carried out using a Pt commonplace in 10% aqua regia starting from 0 to 10 ppm.

### Research of Pt-Exos in vitro cytotoxicity

The therapeutic properties of Pt-Exos as antiproliferative and cytotoxic brokers in several cell cultures have been first decided by utilizing the Blue Cell Viability assay (Promega, United States) as beforehand 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 completely different situations. Strategy 1: cells have been incubated solely with the Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein). Strategy 2: cells have been handled with Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein) containing NH4Cl, and strategy 3: cells have been handled with the Pt-ExoshpMSCs (0.156 µg/µg of exosomal protein) with NH3 inside them.

In a special experiment, 4; 2; 1; 0.5; 0.25; 0.125 µg of exosomes/100 µL loaded with completely different quantity of PtNPs (0.560 and 0.86 µg of Pt/µg of exosomal protein) have been incubated with the cells throughout 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 outcomes are expressed as cell viability proportion, contemplating as 100% the non-treated cells. Cell cultures incubated with the very best dose of exosomes have been additionally noticed by inverted typical microscopy (Olympus IX81) with a purpose to consider their morphology. All of the experiments have been carried out in triplicate.

### In vitro cell demise analysis

To review in additional element the effectivity of Pt-Exos as an antiproliferative and a cytotoxic device, hpMSCs have been seeded onto a 96-well plate (5000 cells/properly). After 24 h in tradition, 4 and a couple of µg of exosomes/100 µL have been added (each from 5x-Pt-ExoshpMSCs and 10x-Pt-ExoshpMSCs). Then, after 48 h, cell tradition media was discarded and cells have been washed twice with PBS. The cytotoxicity produced by the Pt-Exos loaded with completely different quantities of PtNPs was decided with the LIVE/DEAD equipment (Thermo Fisher Scientific, USA) utilizing an inverted fluorescence microscope (Olympus IX81) and following producer directions.

The selectivity and the effectivity of Pt-Exos as therapeutic vectors have been quantified by stream cytometry (FACSAria BD cytometer, BD Bioscience). To find out the impact of the Pt-Exos on the proliferation price of the completely different cell sorts, the distribution of the cell-cycle phases after the incubation of the exosomes was assessed. First, cells have been seeded onto 6-well plates at a density of 250,000 cells/properly. After 24 h, 2 µg of 10x-Pt-ExoshpMSCs and 10x-Pt-ExosU251−MG have been incubated with each hpMSCs and U251-MG cells throughout 48 h. Then, cells have been trypsinized and washed twice with PBS (1500g, 5 min). The ultimate cell pellet was suspended in 200 µL of PBS and was fastened with 70% ice-cold ethanol and maintained at 4 °C in these resolution for twenty-four h extra earlier than being analyzed underneath the stream cytometer. DNA staining was carried out by including RNase A and propidium iodide to the cell resolution. Management samples (non-treated cells and cells handled with the exosomes however within the absence of PtNPs inside them) have been additionally run to know the usual distribution of cells cycles within the cell strains assayed.

The evaluation of cell apoptosis and necrosis was developed by way of a double-staining with Annexin V-FITC (5 µL) and propidium iodide (5 µL) throughout 15 min previous to their evaluation by stream cytometry (FACSARIA BD gear and FACSDIVA BD software program).

### In vivo experiments

The procedures carried out on this examine have been beforehand authorized by the Undertaking License PI07/21 and PI43/21 by the Ethic Committee for Animal Experiments from the College of Zaragoza (Comisión Ética Asesora para la Experimentación Animal de la Universidad de Zaragoza). Mice have been fed advert libitum and their upkeep and care underneath particular pathogen-free situations have been carried out accordingly with the Spanish Coverage for Animal Safety RD53/2013 and the European Union Directive 2010/63 relating to the safety of animals destined to experimental and different scientific functions.

On this examine, six-to eight-week previous feminine BALB/c nu/nu mice (Envigo) have been used. All of the animals have been maintained seven days underneath quarantine as quickly as they arrived to the animal amenities and earlier than beginning the experiments. For the induction of the xenograft tumor, animals acquired a subcutaneous injection of seven × 106 U251-MG cells in 200 µL of saline. To guage the potential weight reduction or ache signs, animals have been weighed and monitored each day. Tumor sizes have been measured with a caliper each two days. The manipulation of the animals was all the time carried out underneath sterile situations in a hood.

After 11 days of tumor implantation, 100 µg of Pt-Exos have been each, IV (within the tail vein) and IT, administered. Alternatively, cisplatin (a medical authorized drug) was administered as optimistic management to match the effectiveness of the Pt-Exos based mostly remedy. The animal teams included within the examine are proven in Further file 1: Desk S1.

With the intention to evaluate the therapeutic impact produced by cisplatin and the Pt-Exos, the administered dose was calculated with a purpose to inject the identical quantity of Pt:

$$0.86,textugfracPtug,exosomecdot 100,textug,exosomes,administered=86,textug,of,Pt,administered$$

Considering the Pt current within the cisplatin molecule:

$$86,textug,of Pt cdot frac300.01,textug,molecule78,textug,Pt=330,textug , of , cisplatin, administered$$

Within the therapeutic examine, as soon as the management animals archive the utmost tumor dimension authorized within the Moral Committee, the experiment was completed and the animals have been sacrificed. Euthanasia of animals was carried out by CO2 inhalation. Chosen organs have been collected from every animal for histopathological evaluation and to find out the quantity of Pt delivered as a consequence of remedy. For histopathological evaluation the samples have been fastened in 4% paraformaldehyde (Alfa Aesar) for twenty-four h, adopted by chilly 70% ethanol. The experimental plan is included in Fig. 1.

Tissue samples have been then embedded in paraffin and three-micrometer sections have been stained with H-E. Immunohistochemical evaluation was carried out by using human Ki-67 antibody (Agilent) with the automated immunostaining platform Autostainer Hyperlink (Dako). Sections (4–5 um) have been dewaxed and rehydrated with dH2O and antigen retrieval was carried out by warmth at pH = 7 throughout 20 min. Tumor samples have been incubated with major Ki-67 antihuman antibody for 20 min adopted by the visualization system conjugated with horseradish peroxidase (Flex/HRP system Agilent). Hematoxylin staining was used as distinction. Lastly, sections have been dehydrated and completely mounted for his or her visualization underneath a standard inverted microscope (Olympus IX81). To acquire the proportion of optimistic cells, Ki-67 expression was assessed in all mice teams by counting optimistic and unfavourable cells in three acquisition photographs from all animals from every group utilizing ImageJ software program.

For the biodistribution evaluation, after 11 days of tumor implantation cisplatin or Pt-ExosU251−MG have been injected within the tail vein and animals have been sacrificed after 24 and 96 h injection. Each teams acquired a complete quantity of 86 µg Pt. Within the management group tumors have been induced nevertheless didn’t obtain any Pt remedy. Pancreas, spleen, kidneys, liver, lungs, mind and tumor have been collected, weighted and digested in an answer of aqua Regia throughout 7 days at room temperature. To guage platinum accumulation; the options have been diluted (1/100) in miliQ H2O and whole quantity of the metallic inside the tissues was decided by ICP-MS (Perkin Elmer Elan DRC-e) within the Chemical Evaluation Service from the College of Zaragoza.

### Statistical evaluation

All the outcomes are expressed as imply ± SD. Statistical evaluation of the info and the numerous variations among the many means have been analyzed by two-way evaluation of variance (ANOVA) for a number of comparisons by Dunnett’s a number of comparisons check (GraphPad Software program). Statistically vital variations have been expressed as follows: *p < 0.05; **p < 0.01; ***p < 0.0001 and ****p < 0.00001.