Chloroquine as a promising adjuvant therapy for type 1 Diabetes Mellitus

Chloroquine as a promising adjuvant therapy for type 1 Diabetes Mellitus

Preparation of CQ NPsCQ was obtained commercially (Sigma-Aldrich Co. cat # C1386, lote # 081M1611V) and the CQ incorporated into PLA NPs was prepared according to the methods of Lima et al.40.Briefly, CQ NPs were prepared using the emulsification-solvent evaporation method. The procedure was performed by mixing 0.4 mL of 0.5 M aqueous NaHCO3 (pH = 8.4) containing 1.25% v/v with 10 mL of organic phase dichloromethane containing PLA (0.25% v/v) to obtain a CQ:PLA ratio of 1:5 v/v. Then, the vial was vortexed for three cycles of 10 s at 1 min intervals. After, the supernatant (NaHCO3 solution) was removed and 4 mL of the organic phase containing CQ and PLA was injected at 1.0 mL/min in the aqueous phase (16 mL) containing the surfactant, poloxamer 407 (0.75% v/v), under magnetic stirring. The phases were filtered, and emulsification was produced under shaking via Ultra-turrax equipment (IKA Labortechnik, Staufen, Germany) with evaporation overnight. The samples were stored at 8 °C and the size and zeta potential were measured after 24 h41.Study populationOverall, 25 patients with T1DM aged between 10 and 16 years (T1DM group) were recruited from the Pediatric Endocrinology Unit, Onofre Lopes Universitary Hospital of the Federal University of Rio Grande do Norte (UFRN), Natal, RN, Brazil. All patients with T1DM were on continuous insulin. T1DM diagnosis was in accordance with the ADA criteria with fasting blood glucose ≥ 126 mg/dL and HbA1C ≥ 6.5%1,3. Glycemic controls were evaluated by measuring HbA1c in whole blood as well as the serum glucose concentration. All tests were performed using Wiener kits, according to the manufacturer’s instructions, using the biochemical analyzer CMD-800 (Wiener Laboratories, Rosario, Argentina).In addition, 25 unrelated subjects (normoglycemic group, NG) with no previous diagnosis of T1DM, a fasting serum glucose of ≤ 99 mg/dL, and in the same age range were recruited from local public schools in Natal, RN, Brazil. The study was conducted in accordance with the guidelines set by the Ethics in Research Committee of the Federal University of Rio Grande do Norte, which complies with the Declaration of Helsinki, being approved by Research Ethics Committee of the Onofre Lopes Hospital of the UFRN under protocol number 704.310. Written informed consent was obtained from all adult subjects and parents or legal guardians of underaged patients and normoglycemic individuals. The exclusion criteria were pregnancy, hypertension, the presence of inflammatory diseases, and recent infections. After assessing medical history, fasting blood samples were obtained from all subjects for biochemical analyses, cell isolation, cell culturing, and extraction of total RNA.Isolation and culture of PBMCsPBMCs were obtained from the NG and T1DM groups by density gradient, in which blood was added to Histopaque® (Sigma-Aldrich, St Louis, MO, USA; density gradient 1.077 g/mL) and centrifuged to 400 × g for 30 min. After centrifugation the cells were resuspended in 2 mL RPMI 1,640 medium supplemented with 0.29 g/L L-glutamine, 2 g/L sodium bicarbonate, 25 mM/L HEPES, 1% antibiotic–antimycotic, and 10% serum. The cells were counted in a Neubauer chamber and checked for viability using trypan blue dye. After counting, the cells were diluted to obtain the required amounts for the experiments.PBMC viability after treatment with CQ and CQ NPsThe viability of the PBMCs of the T1DM group was assessed by MTT (Sigma-Aldrich, St Louis, MO, USA). Cells were cultured in 96-well plates with 1 × 104 cells/well, exposed to CQ and CQ NP concentrations of 5, 10, 25, 50, 100, and 200 μM, and maintained in a CO2 stove (5%) at 37 °C for 48 h. Then, 5 mg/mL MTT was added to each well and the plate was incubated in a CO2 oven (5%) at 37 °C for 4 h. After, 100 μL of 10% sodium dodecyl sulfate in 0.001 N hydrochloric acid was added for dissolution of the crystals. The plate was then read in a microplate spectrophotometer at 540 nm (Biotek®, Epoch model, Winooski, Vermont, USA). The experiment was carried out in triplicate. Cell viability was defined in comparison to untreated controls. The relative cell viability (%) in the sample-treated wells with respect to the control wells was estimated by Eq. 1:$${text{% Cell Viability = }}frac{{{text{(Tested)}} times {100}}}{{text{(Control)}}}$$In which tested and control are the absorbance of treated sample and control sample, respectively.To determine the CC50 values, nonlinear regression of concentration–response curves was determined. The CC50 was defined as the concentration that reduced cell viability by 50% compared to the untreated controls.T1DM PBMC treatment with CQ and CQ NPsT1DM PBMC suspensions (1 × 105 cells/well) were seeded in 24-well flat-bottomed culture plates (Corning Incorporated, Corning, New York, USA). The cells were subjected to three different conditions; no treatment (NT), treatment with CQ (CQ), and treatment with CQ NPs (CQ NP). Cells of the NG group were treated with culture medium only. The plates were maintained in a CO2 incubator (5%) at 37 °C for 24 and 48 h. Cells were then resuspended and collected at the end of each incubation period, then centrifuged at 500×g for 3 min to obtain the cells, and the pellet was used to obtain total RNA.Real-time quantitative polymerase chain reaction (qPCR) analysisTotal RNA was extracted from the cell pellet obtained from the T1DM and NG groups using a Promega SV Total RNA Isolation System (Promega Corporation, Madison, WI, USA), according to the manufacturer’s instructions. The concentration and purity of the total RNA were measured using a nanodrop spectrophotometer (NanoDrop ND-1000, Montchanin, DE, USA) at 260 nm.The Integrity of the RNA was determined by agarose gel electrophoresis (2% agarose/3-[N-morpholino] propanesulfonic acid) using aliquots of total RNA (approximately 10 μL). The agarose gel was stained with GelRed™ (Uniscience, São Paulo, SP, Brazil), revealing the presence of two sharp bands at approximately 5 and 1.8 Kb that corresponded to ribosomal RNA 28S and 18S, respectively.Expression levels of IL1B, IL12, IFNG, TNFA, and IL10 were evaluated via real-time qPCR. The normalizing reference gene was beta-actin (ACTB), which showed the greatest stability among the glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine phosphoribosyltransferase 1, and beta-2-microglobulin genes after analyses by GENORM™ (v.3.44) and NORMFINDER software42.All gene expression analyses were performed in a 7,500 Fast Real-Time PCR System (Applied Biosystems, CA, USA). Reactions were carried out in duplicate using a SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR kit (Life Technologies Corporation, Carlsbad, CA, USA) with a total volume of 12 μL containing approximately 10 ng of total RNA. Real-time PCR cycling conditions were as follows: 55 °C for 30 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.Relative expression was calculated using the 2−ΔΔCt method43. Significance was determined via the comparison of ΔCt values for each target gene, and results are presented as the fold‐change of the NG group mean values normalized to ACTB.Statistical analysisAll data were expressed as mean ± standard deviation. The distribution of the variables was analyzed using the Kolmogorov–Smirnov test. Variables with normal distributions were subjected to Student’s t-test and ANOVA analysis, followed by comparison with Tukey’s test. Data were analyzed in GraphPad Prism v.6.0 (GraphPad Software, Inc., San Diego, CA, USA). Statistically significant results were obtained at p < 0.05.

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