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HomeNanotechnologyFunctionalized nanoparticles with monocyte membranes and rapamycin obtain synergistic chemoimmunotherapy for reperfusion-induced...

Functionalized nanoparticles with monocyte membranes and rapamycin obtain synergistic chemoimmunotherapy for reperfusion-induced damage in ischemic stroke | Journal of Nanobiotechnology

Medicine and supplies

Poly (lactic-co-glycolic) acid (PLGA, MW 900000, 50:50) and RAP have been bought from Chengdu Good Alumni Biotechnology Co., Ltd. (Sichuan, China). 1,1ʹ-Dioctadecyl-3,3,3ʹ,3ʹ-tetramethylinddodicarbocyanine perchlorate (DiD) was bought from Biotium Inc. (Fremont,US). Opti-mum reducing temperature compound (OCT), 2,3,5-triphenyltetrazolium chloride (TTC) and 4,6-diamidino-2-phenylindole (DAPI) have been bought from Beijing Solarbio Science & Expertise Co., Ltd. (Beijing, China). An enhanced BCA protein assay package, membrane and cytosol protein extraction package and PMSF have been bought from Beyotime Institute of Biotechnology (Jiangsu, China). The rat IL-4 ELISA package and rat IL-10 ELISA package have been provided by MultiScience (Lianke) Biotech Co., Ltd. (Zhejiang, China). Polycarbonate porous membranes have been provided by Whatman Worldwide Ltd. (Maidstone, Kent, UK). Ultrapure water with a resistivity of 18.2 MΩ cm was used all through the experiments.

Preparation of the monocyte membranes

Monocyte membranes derived from RAW264.7 cells have been extracted as beforehand described with slight modification [19, 31]. The cells have been cultured in DMEM (Gibco) with 10% FBS and 1% penicillin–streptomycin (Gibco). Monocyte membranes have been obtained utilizing a Membrane and Cytosol Protein Extraction Equipment. Three bottles of cells have been collected and washed with PBS thrice (2500 rpm for five min every) [32]. Then, 990 mL membrane protein extraction reagent A and 10 mL of PMSF (Beyotime) have been added to an ice bathtub for 15 min. After that, the combination was first frozen at − 80 ℃ and thawed at room temperature, and the cells have been subjected to three–5 freeze–thaw cycles [31, 33]. The obtained combination was centrifuged at 1500 rpm (4 ℃, 10 min) to take away cytoplasm and varied organelles. Subsequent, the cell membranes have been obtained at 12,000 rpm (4 ℃, 30 min) [16]. An enhanced BCA protein assay package was used to investigate the whole protein content material within the obtained monocyte membranes. These membranes (named McM vesicles) have been saved at − 80 ℃ for later use.

Fabrication of Rapamycin-loaded PLGA nanoparticles (RNPs)

PLGA and rapamycin (RAP) have been dissolved in N, N-Dimethylformamide (DMF), and 40 mg/L and 10 mg/L options have been generated, respectively. RNPs have been obtained by the nanoprecipitation methodology as beforehand described, with a minor modification [33, 34]. Then, 250 mL of PLGA and 200 mL of RAP have been added in 550 mL of DMF (whole 1 mL combination). The combination was added dropwise to three mL ultrapure water with light stirring, after which transferred to a dialysis bag (molecular weight cutoff (MWCO) of 3500 Da) to take away free RAP and DMF. The RNPs answer was saved at 4 ℃. To acquire fluorescently labeled NPs, PLGA was combined along with 1,1ʹ-dioctadecyl-3,3,3ʹ,3ʹ-tetramethylindodicarbocyanine (DiD) dissolved in N,N-Dimethylformamide (DMF) to type DiDNPs. For fluorescent imaging experiments, 0.1 wt% DiD (excitation = 644 nm, emission = 665 nm) was encapsulated into PLGA cores to type DiDNPs [35].

Fabrication of McM camouflaged RNPs (McM/RNPs)

Membrane-coated RNPs have been fabricated by a beforehand reported sonication [33] and coextrusion methodology [16]. Briefly, collected McM vesicles and RNPs have been combined at a membrane protein-to-polymer ratio of 1:1 (w/w). Subsequent, the combination was sonicated for five min in a FS30D bathtub sonicator with a frequency of 42 kHz and an influence of 100 W. Then, McM/RNPs have been obtained through the use of the Avestin mini extruder (Avestin, LF-1, Canada) extruded 10 occasions by a 200 nm polycarbonate porous membrane. McM and DiDNPs have been combined and McM/DiDNPs have been formulated by the coextrusion methodology.

Nanoparticle characterization

RNPs, McM vesicles, and McM/RNPs have been measured for measurement and zeta potentials with dynamic mild scattering (DLS) utilizing a Malvern ZS 90 Zetasizer. The nanoparticle morphology was examined by transmission electron microscopy (TEM) at 200 kV (JEM-2100F, JEOL, Japan). TEM was carried out by depositing the particles onto a 300-mesh glow-discharged carbon-coated grid (Beijing Zhongjingkeyi Expertise Co., Ltd.) and detrimental staining with 1 wt% phosphotungstic acid.

Drug loading and encapsulation effectivity of nanoparticles

Briefly, 1 mg of RAP was combined with 10 mg PLGA copolymer at a weight ratio of 1:10 and dissolved in 1 mL of DMF. To calculate the loading effectivity of RAP, RNPs lyophilized energy was dissolved in DMF, and the absorbance was measured utilizing an ultraviolet spectrophotometer (DU730, Beckman Coulter) at 280 nm. In response to the pre-established commonplace curve of RAP in DMF, the concentrations of the usual curve have been 0, 2, 4, 8, 16 and 32 mg/mL. The drug loading effectivity (LE) and drug encapsulation effectivity (EE) have been calculated by Eq. (1) and (2), respectively [16, 36].

$$mathrm{LE }(mathrm{%})=frac{Weight of RAP in lyophilized powder }{Weight of PLGA+Weight of RAP in lyophilized powder} occasions 100mathrm{%}$$


$$mathrm{EE }(mathrm{%})=frac{Weight of RAP in lyophilized energy}{Weight of added RAP} occasions 100mathrm{%}$$


SDS-PAGE gel and western blotting

Samples containing monocytes, McM, and McM/RNPs have been denatured and loaded into a ten% SDS-polyvinylidene gel. The membrane proteins of the McM vesicles and McM/RNPs have been extracted by Cell lysis buffer for Western blotting (Beyotime Institute of Biotechnology) and centrifuged at 12,000 rpm for 20 min at 4 ℃. Protein quantification was carried out utilizing a BCA Protein Assay package (Beyotime Institute of Biotechnology). All of the extracted proteins have been run on a Bis–Tris 10-well mini-gel in operating buffer utilizing a Bio-Rad electrophoresis system at a continuing of 80 V for 30 min after which at 120 V for 90 min. Subsequent, the ensuing polyacrylamide gel was stained with Coomassie blue for visualization.

Moreover, the integrin a4, integrin β1, CD47, and b-actin contents in monocytes, McM, McM/RNPs, and RNPs have been decided by Western blot evaluation. Samples have been subjected to 10% SDS-PAGE and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA). The membranes have been blocked with 5% nonfat milk in TBST buffer (10 mM Tris–HCl, 100 mM NaCl, pH 7.4, 0.1% Tween-20) at 25 ℃ for 1 h. Then, the membranes have been incubated with main antibodies: a4 (1:5000 dilution, CST), β1 (1:5000 dilution, CST), CD47 (1:1000 dilution, ABclonal) and β-actin (1:5000 dilution, GeneTex) at 4 ℃ in a single day within the fridge. After the membranes have been washed thrice with TBST, they have been additional incubated with horseradish peroxidase-conjugated home anti-mouse secondary antibody (1:2000 dilution, Cell Signaling) or goat anti-rabbit secondary antibody (1:2000 dilution, Cell Signaling) at room temperature for 1 h. Protein bands have been visualized by the improved chemiluminescence methodology utilizing a ChemiDoc MP imaging system (Bio-Rad, USA).

In vitro launch of nanoparticles

The drug of RAP launch from RNPs and McM/RNPs have been studied utilizing a dialysis methodology. Briefly, RAPNP and McM/RNP options (2 mg/mL, 3 mL every) have been added to disposable dialysis luggage (MWCO: 3500 Da, Thermo Scientific). The dialysis luggage have been then immersed in 12 mL of PBS answer (Launch medium, pH 7.4) at room temperature. Three unbiased replicates have been used for every pattern. Two milliliters of launch medium was collected for evaluation at completely different time intervals (1 h, 2 h, 4 h, 8 h, 12 h, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days and 9 days) and changed with an equal quantity of contemporary PBS at room temperature. The cumulative quantity of RAP launched was quantified by a UV/Vis spectrophotometer (DU 730, Beckman Coulter) at 280 nm.

Mobile uptake

To judge the consequences of McM camouflaging on phagocytosis discount by macrophages, experiments have been carried out utilizing RAW264.7 macrophage cells. The internalization of DiDNPs and McM/DiDNPs by macrophages cells was evaluated by CLSM and FACS measurements. Briefly, RAW264.7 macrophage cells have been seeded in 24-well plates at a density of 1 × 105 cells per properly in 500 μL of DMEM supplemented with 10% FBS and cultured for 12 h. Then, 150 μg of DiDNPs or McM/DiDNPs have been added to every properly. After coincubation for 0.5, 1, 2 and 4 h, the nuclei have been stained with DAPI for CLSM imaging. Cells have been collected for quantification by FACS evaluation.

Quantification of NPs uptake in macrophages was measured by movement cytometry (BD, USA). Macrophages have been seeded in 12-well plate with a density of 1 × 106 cells/properly. After 12 h incubation in 37 °C incubator containing 5% CO2, 100 μL (2.5 mg/mL) of DiDNPs or McM/DiDNPs options have been added into every properly, then the cells have been incubated with the options for 0.5, 1, 2 and 4 h, respectively. After incubation, the cells have been washed for thrice with 1× PBS and digested with trypsin for assortment. Then, the cells have been washed with PBS twice earlier than centrifuge at 1500 rpm for five min. Lastly, the cells have been resuspended in 1 mL of 1× PBS. Three replicates have been adopted for every group and 1 × 105 cells have been analyzed in every pattern.To substantiate that the nanoparticles have been engulfed by the macrophages. RAW264.7 macrophage cells have been seeded in 24-well plates at a density of 1 × 105 cells per properly in 500 μL of DMEM supplemented with 10% FBS and cultured for 12 h. After then, 150 μg of DiDNPs or McM/DiDNPs have been added to every properly. After incubate 4 h, the medium was modified to DMEM containing Lyso-Tracker Inexperienced. After coincubation, the nuclei have been stained with Hoechst for 10 min. Then, the cells have been washed twice with 1× PBS and imaged with a laser confocal microscope.

In vitro Transwell assay

Within the Transwell fashions, HUVECs have been inoculated into the higher chamber with a diameter of 0.40 mm in a 24-well plate (1 × 105 per properly), whereas medium was added to the decrease chamber and stimulated with lipopolysaccharides (LPS, 10 mg/mL). When the ECs grew to become a monolayer, LPS was added for 12 h, after which the brand new medium was modified and cocultured with McM/DiDNPs at completely different occasions. The medium within the decrease chamber was collected, and the fluorescence depth was measured with an F-4700 fluorescence spectrophotometer (Japan).

Monocyte adhesion assay

Human umbilical vascular endothelial cells (HUVECs) grown to confluency in 24-well plates have been stimulated with LPS (10 mg/mL) for 12 h and washed with PBS. THP-1 cells (Cell Financial institution of Chinese language Academy of Sciences) have been labeled with Hoechst. Subsequent, the labelled THP-1 cells have been overlaid on the HUVECs and incubated for two h. After washing, the numbers of THP-1 cells adhering to the HUVECs monolayer in every properly have been examined by a fluorescence microscope (Olympus Company, Japan). Lastly, the variety of THP-1 cells was counted by ImageJ software program.

Animal experiments

All experiments have been carried out utilizing male Sprague–Dawley rats (SD, 280–320 g), bought from Hunan SJA Laboratory Animal Co. Ltd. (Hunan, China). Procedures that used animals have been reviewed and permitted by the Tumor Hospital of Chongqing College Ethics Committee and in accordance with the Information for the Care and Use of Laboratory Animals (Institute for Laboratory Animal Analysis, Nationwide Analysis Council, Washington, DC: Nationwide Academy Press, 1996). The rats have been housed below alternating 12 h mild/darkish circumstances and allowed free entry to water and meals earlier than all experiments. The rats have been divided into 5 teams: 5% glucose, RAP, RNPs, McM, or McM/RNPs.

Transient center cerebral artery occlusion (tMCAO) fashions have been generated in keeping with strategies that have been lately reported [37,38,39]. Every SD rat was anesthetized with 5% isoflurane (Aerrane, Baxter, Deerfield, IL) in a combination of 70% nitrous oxide and 30% oxygen. Isoflurane was then maintained at 1.5%. Throughout the procedures, the physique temperature of the rats was monitored at 37.0 ± 0.5 ℃. Rats have been positioned within the supine place and the proper frequent carotid artery (CCA), exterior carotid artery (ECA), and inside carotid artery (ICA) have been fastidiously uncovered and dissected from the encircling tissue. Then, a small gap within the ECA was made utilizing Vanes-style spring scissors. A silicon-coated monofilament (Equipped by Guangzhou Jialing Biotechnology Co., Ltd. Guangdong, China) inserted into the ICA through the CCA, 8–10 mm away from the bifurcation, blocked the start line of the center cerebral artery. Profitable MCA occlusion was confirmed by a discount in rCBF by over 80%. The occlusion lasted 90 min, and the monofilament was withdrawn to permit for reperfusion.

Lengthy-term circulation operate of nanoparticles in vivo

DiDNPs and McM/DiDNPs have been administered in rats through the tail vein at a nanoparticle dosage of two mg/kg. 1 mL of 0.09% physiological saline was added to anticoagulation tube. At completely different time intervals (1, 2, 3, 4, 5 and 6 days), the tail of the rats have been pierced, then 20 μL of entire blood was taken and dissolved in an anticoagulation tubes. The fluorescence depth of the remaining nanoparticles within the blood have been measured by fluorescence microplate reader.

Ex vivo imaging

Rats with ischemic stroke have been used for mind focusing on analysis nanoparticles [40]. Briefly, DiDNPs and McM/DiDNPs have been administered through the tail vein at a nanoparticle dosage of two mg/kg. After 24 h, the rats have been euthanized and perfused with precooled 0.09% physiological saline to take away the blood and unbound nanoparticles. For ex vivo near-infrared fluorescence imaging, photos have been captured utilizing a near-infrared fluorescence in vivo imaging system (IVIS Lumina III, Perkin Elmer, USA) and quantitatively analyzed utilizing Dwelling Picture 5.0 software program. The organs, together with the mind, coronary heart, liver, spleen, lung, and kidney have been subsequently fastened and used for histological evaluation in the principle organs and the buildup of DiDNPs or McM/DiDNPs in stroke lesions [41].

2,3,5-Triphenyltetrazolium chloride (TTC) staining

To determine the first infarction, rats (n = 15) chosen randomly from ischemia within the 5% glucose, RAP, RNPs, McM, and McM/RNPs teams (3 rats per group) have been sacrificed 7 days after MCAO below deep anesthesia as above and a couple of mm thick coronal mind sections have been ready and immersed in 2% TTC (Solarbio, China) in saline for 20 min at 37 °C and glued for 30 min in 4% paraformaldehyde [42].

Immunofluorescence staining

Immunofluorescence was carried out as beforehand described [43,44,45,46]. Frozen mind Sects. (10 mm) have been ready utilizing a cryostat (Leica, CM1900) in keeping with commonplace procedures. Briefly, sections have been preincubated with 0.5% Triton X-100 (v/v) and 5% BSA in PBS for 10 min, adopted by blocking in 5% bovine serum albumin (BSA) for 1 h at room temperature. Then, sections have been incubated in a single day at 4 ℃ with main antibodies diluted in QuickBlock™ Main Antibody Dilution Buffer for Immunol Staining (Equipped by Beyotime). The next main antibodies have been used: mouse anti-VCAM-1 (1:50, Santa Cruz, CA, USA), mouse anti-ICAM-1 (1:50, Santa Cruz, CA, USA), mouse anti-NeuN (1:200, Millipore, Bedford, USA), rabbit anti-GFAP (1:200, Abclonal, Wuhan, China), mouse anti-rat CD11B (1:150, Millipore, Bedford, USA), and mouse anti-CD68 (1:100, Abcam, USA). After washing the unbound antibody thrice, the cells have been washed in PBST (0.05% Tween-20 in PBS). The sections have been incubated with secondary antibodies for 1 h at room temperature. The secondary antibodies have been Alexa Fluor® 555 Donkey polyclonal secondary antibody to rabbit IgG-H&L (1:500, Abcam, USA) or Alexa Fluor® 647 Donkey polyclonal secondary antibody to mouse IgG-H&L (1:500, Abcam, USA). The cells have been washed thrice with PBST and counterstained for nuclei with DAPI (Solarbio, Beijing, China). Antifade mounting medium (Beyotime, Jiangshu, China) was used, and fluorescent photos have been obtained utilizing a confocal laser scanning microscope (TCS SP8, Leica, Germany).

Neurological purposeful evaluation

Rat behavioral testing and scoring have been assessed by two individuals following the directions adopted from Bederson [47, 48]. Neurological scores have been decided 3 and seven days after MCAO, the efficient neurological rating of every rat was the common of neurological rating from the 2 observers. Bederson scores have been used to judge world neurological operate as beforehand described [49]. Briefly, the rats have been suspended by the tail 20 cm above the ground. The animals have been scored primarily based on the signs of the rats: 0 factors, rats behave usually; 1 level, rats can not totally stretch their left entrance legs; 2 factors, rats flip round right into a circle; 3 factors, rats fall right down to the left aspect; and 4 factors, rats can not transfer by themselves, dropping their consciousness.

Full blood depend and medical chemistry

Blood was collected in EDTA spray-coated tubes and instantly analyzed for hematological parameters by an automatic hematology analyzer (Sysmex KX-21, Sysmex Co., Japan), similar to crimson blood cells, hemoglobin (HGB), lymphocytes, monocytes and neutrophils. The plasma concentrations of aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (UREA), and creatinine (CREA) have been quantified by an automatic analyzer platform (Roche Cobas C501, Roche Co., Switzerland).

Statistical evaluation

GraphPad Prism software program (GraphPad, USA) was used for statistical evaluation. Two-tailed Pupil’s t check was used for comparability between two teams. Comparisons amongst three or extra teams have been carried out utilizing one-way ANOVA. Statistical significance ranges have been set to * p < 0.05, ** p < 0.01, *** p < 0.001 and **** p < 0.0001, with all information displayed because the imply ± SD.



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