To determine chondrocyte viability upon co-incubation with NLC, a standard method to measure cytotoxicity, AlarmarBlue® (Thermo Fischer Scientific, Waltham, MA, USA.), was used. In this assay, a redox indicator dye, resazurin, which changes color and fluoresces in response to chemical reduction due to cell growth, evaluated the metabolic activity of cells and in turn, determined the concentration of viable chondrocytes in each well. Fluorescence was detected using Fluostar Optimaequipment (BGM LABTECH Inc. Cary, NC, USA). Cell viability was expressed as the fluorescence measurement with test cells relative to untreated control cells. Primary fibroblasts from healthy patients were seeded in 96 well plates at a density of 4000 cells per well for 24 h at 37 °C. From a stock solution of 500 mg/mL of NLC, 100 µL was used to prepare serial dilutions and was co-incubated with cells for 24 h at 37 °C. After the cells were washed with PBS, 10 µl of AlamarBlue® reagent was added to each well to rech a final volume of 100 µL. Following a 3 h incubation period at 37 °C, the plate was removed from the incubator and the fluorescence was measured with an excitation wavelength of 544 nm and emission wavelength of 590 nm (FLUOstar OPTIMA, BGM LABTECH Inc. Cary, NC, USA).
Internalization Studies of NLC in TC28a2 Chondrocytes and Pathological Fibroblasts from MPS IVA Patients
The capacity of internalization of the optimized NLC containing elosulfase alfa was investigated in the TC28a2 chondrocyte cell line using confocal microscopy and transmission electron microscopy (TEM) and in pathological fibroblasts from Morquio A patients using TEM. Additionally, the enzyme activity of cells after co-incubation with NLCs was studied.
For confocal microscopy, the NLC containing the elosulfase alfa (using different dilutions of NLC formulation from the 200 mg/mL suspension) was stained previously with DiD (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate salt) with the purpose of staining lipids in red; this was then incubated in the co-culture of 100,000 cells in 24 well plates on glass covers at 37 °C or 4 °C for 24 h (HERA cell 150, Thermo Fisher Scientific). After that time, cells were stained with calcein (Fluorexone; Sigma-Aldrich) by incubating cells for 20 min at 37 °C and 4 °C. The co-incubation of NLC in cell cultures were performed at different times, i.e., 1 h, 2 h, or 24 h. Upon NLC exposure, NLC-incorporated cells were fixed for confocal examination using the fluorescence microscope Leica TCS-SP8 (Leica Microsystem, Buffalo Grove, IL, USA).
The cells were imaged using the following resolution conditions: 1024 × 1024 pixels and image size of 184.82 × 184.82 µm. Z-stacks were recorded at 0.3 µm spacing using an objective HC PL APO CS2 63x/1.40 oil, pinhole: 95.3 µm. The cell samples with or without NLC were excited at 638 nm for red dye and 488 nm for green dye, and the emission was filtered through a band-pass filter (646–778 nm and 495–564 nm, respectively).
The interaction of NLC and elosulfase alfa with cells was visualized using TEM with a JEOL JEM 1011 microscope (JEOL Ltd., Tokyo, Japan). TC28a2 chondrocytes or pathological fibroblasts samples were grown onto Thermanox slides (6well plates) and co-incubated for 1 h at 37 °C with NLC (100 µL from 200 mg/mL). After 24 h, cells were centrifuged and fixed with 2.5% of glutaraldehyde in 0.2 M phosphate buffer overnight. The samples were pots-fixed with 1% osmium tetroxide in 0.05 M cacodylate buffer for 1 h and sequentially dehydrated with 50%, 70%, 90%, and 100% methanol. Finally, cells were embedded in Epon 812 resin and sections that were 5 µm thick were cut. Sections were stained with uranyl acetate and lead citrate solution.
Electrophoretic Identification of Elosulfase Alpha in Cells and Quantification by MALDI-TOF Analysis
To analyze the proteins, cells were detached with trypsin, resuspended in the culture medium, centrifuged at 1000 rpm, and washed twice with purified water to remove all remaining proteins. Finally, cells were resuspended in 500 μL of milli-Q water and lysed using ultrasound. Samples were then centrifuged at 13,000 rpm for 10 min at 4 °C to separate the protein extract from the broken cell membranes.
An amount of 100 µg of protein extract as loaded onto a 10% SDS-PAGE gel. The protein band was detected by Sypro-Ruby fluorescent staining (Lonza, Rottenstrass, Switzerland), excised, and processed by manual tryptic digestion. Peptides were extracted by carrying out three 20 min incubations in 40 μL of 60% acetonitrile dissolved in 0.5% HCOOH. The resulting peptide extracts were pooled, concentrated in a SpeedVac, and stored at −20 °C.
Four micrograms of digested peptides was separated using reverse phase chromatography. gradient (micro liquid chromatography system; Eksigent Technologies nanoLC 400, SCIEX, coupled to high-speed Triple TOF 6600 mass spectrometer (ABSciex, Foster City, CA, USA) with a microflow source). The analytical column used for analysis was the silica-based reversed-phase column Chrom XP C18 150 × 0.30 mm with 3 mm particle size and 120 Å pore size (Eksigent, ABSciex, Woodlands Central Indus. Estate, Singapore). The trap column was the YMC-TRIART C18 (YMC Technologies, Teknokroma, Barcelona, Spain) with a 3 mm particle size and 120 Å pore size, switched online with the analytical column. The loading pump delivered a solution of 0.1% formic acid in water at 10 µL/min. The micro-pump generated a flow-rate of 5 µL/min and was operated under gradient elution conditions, using 0.1% formic acid in water as mobile phase A and 0.1% formic acid in acetonitrile as mobile phase B. The peptides were separated using a 90 min gradient ranging from 2% to 90% mobile phase B (mobile phase A: 2% acetonitrile, 0.1% formic acid; mobile phase B: 100% acetonitrile, 0.1% formic acid).
Data acquisition was performed using the TripleTOF 6600 System (SCIEX, Foster City, CA, USA) via a data-dependent workflow. Source and interface conditions were an ion spray voltage floating (ISVF) of 5500 V, curtain gas (CUR) 25, collision energy (CE) 10, and ion source gas 1 (GS1) 25. The instrument was operated with Analyst TF 1.7.1 software (ABSciex, Woodlands Central Indus. Estate, Singapore). The switching criteria was set to ions greater than a mass-to-charge ratio (m/z) of 350 and smaller than m/z of 1400 with a charge state of 2–5, a mass tolerance of 250 ppm, and an abundance threshold of more than 200 counts (cps). Former target ions were excluded for 15 s. The instrument was automatically calibrated every 4 h using external calibration tryptic peptides from PepCalMix.
Pathological fibroblasts were incubated with chondroitin-6-sulphate (C6S)(Sigma Aldrich), allowing for the quantification of GAGs.
For the quantification of GAG concentrations in cells, 1-9 dimethylmethylene blue (DMB) (Sigma Aldrich) was used.
Pathological fibroblasts, with a total of 100,000 cells, were seeded in well plates and left for 24 h to adhere to the plate surface. Cells were incubated with a concentration of 6.25 mg/dL of C6S to promote the intracellular accumulation. Tree replicates were incubated additionally with 100 μL of solution containing elosulfase alfa-NLC at a concentration of 200 mg/mL (equivalent to 50 ng/mL of free enzyme).
For C6S quantification, resuspended cells were counted in a Neubauer chamber, lysed by ultrasound, and centrifuged at 13,000 rpm to recover the protein extract. Then, 1 mL of DMB was added to 500 μL of supernatant, incubated for 15 min at room temperature, centrifuged for 10 min at 13,000 rpm, and then the supernatant was decanted. Then, 50 μL of 7.5% SDS and 450 μL of purified water were added. The solution was sonicated again and 200 μL was dispensed to a 96 well plate for determination by 595 nm spectrophotometry (EPOCH-2, BioTeK, Swindon ,UK).