Supplies
CAT was bought from Beyotime Biotechnology (Shanghai, China). 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine-conjugated diphenyl-poly(ethylene glycol)2k monomethyl ether-cyclooctyne (DSPE-PEG2k-DBCO) was bought from Ruixi Organic Know-how Co., LTD. (Xi’an, China). α-Helical polypeptide bearing guanidine teams on the aspect chains (PG, polymerization diploma = 102) was synthesized in accordance with our earlier report [54]. Lysotracker Pink and Trizol reagent had been bought from Thermo Fisher Scientific (Massachusetts, USA). BCA protein assay package, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), and 4’,6-diamidino-2-phenylindole (DAPI) had been bought from Alfa Aesar (Shanghai, China). PrimerScript real-time reagent package and SYBR Premix Ex Taq package had been bought from Takara Bio (Qingdao, China). Azide-modified sSDF-1α peptide (N3-KSKPVVLSYR, N3-sSDF-1α) and Cy3-labeled N3-sSDF-1α had been bought from Bankpeptide Biotechnology Co., LTD. (Hefei, China). Major antibodies (anti-Col I, anti-ALP, anti-Runx2, anti-OCN, and anti-OPG) had been bought from Solarbio Life Sciences Co., LTD. (Beijing, China) or Beyotime Biotechnology (Shanghai, China). Secondary antibodies of horseradish peroxidase-conjugated goat anti-mouse IgG and goat anti-rabbit IgG had been bought from Absin Bioscience Co., LTD. (Shanghai, China). APC-labeled anti-CD105, PE-labeled anti-CD73, and Percy/Cy5.5-labeled anti-CD45 had been bought from Elabscience Biotechnology Co., LTD (Wuhan, China). siCkip-1 and adverse management siRNA with scrambled sequence (siScr) had been bought from GenePharma Co., LTD. (Shanghai, China), and their sequences had been listed in Supplementary: Desk S1. All primers had been bought from Sangong Biotech (Shanghai, China), and their sequences had been listed in Supplementary: Desk S2. All the opposite reagents had been bought from Vitality Chemical (Shanghai, China) or Aladdin (Shanghai, China) and used as obtained.
Animals and cells
Feminine C57BL/6 mice (6–8 weeks, 18–20 g) had been bought from Slaccas Experimental Animal Co., LTD. (Shanghai, China) and housed in a selected pathogen-free (SPF) animal lab.
RAW 264.7 cells (mouse monocyte macrophage) had been bought from the American Kind Tradition Assortment (Rockville, MD, USA) and cultured in DMEM containing 10% fetal bovine serum (FBS). Mouse MSCs (bone marrow mesenchymal stem cells) had been bought from Cyagen Biosciences Co., LTD. (Guangzhou, China) and cultured in α-MEM containing 10% FBS.
Mouse femur fracture mannequin
The femur fracture mannequin was established in accordance with a earlier report [43]. Significantly, mice had been anesthetized, and their proper hips, thighs, and knees had been sterilized with povidone-iodine resolution. A 2-cm medial parapatellar incision was created and the patella was dislocated to reveal the femoral condyles. A gap was drilled into the femoral intramedullary canal on the intercondylar notch utilizing a 25-gauge needle to stabilize the upcoming fracture. Instantly after the needle implantation, blunt dissection of muscle was carried out to reveal the midshaft of the femur, and a transverse femoral shaft fracture was then created in the suitable femur of every mouse utilizing a rotary Dremel with a diamond blade attachment. The patella was then decreased, and the incision was closed utilizing 4 − 0 artificial suture. Mice had been allowed to maneuver freely after restoration from anesthesia. On day 5 put up fracture, animals had been imaged with an X-ray imaging system (Faxitron MX-20, Tucson, AZ) to confirm that the mid-diaphyseal fracture in femur had been produced.
Cell membrane isolation
RAW 264.7 cell membrane because the MM was remoted in accordance with a earlier report [62]. Briefly, RAW 264.7 cells on the tradition dish (60 mm in diameter) had been harvested and suspended within the homogenization buffer containing Tris·HCl (pH 7.5, 20 mM), potassium chloride (10 mM), sucrose (75 mM), magnesium chloride (2 mM), and protease/phosphatase inhibitors. The suspension was disrupted with a probe ultrasonic disruptor (JY 92-IIN, Ningbo Scientz, 100 W, sonicate for five s and pause for five s, 10 min, 4 ºC) after which centrifuged (20,000 g, 25 min). The supernatant was centrifuged (100,000 g, 35 min) once more, and the pellet was collected because the RAW 264.7 cell membrane and saved at -80 ºC till use. The protein content material of MM was decided utilizing the BCA protein assay package. For fluorescence microscopy imaging and fluorescence resonance power switch (FRET) evaluation, DiO-stained MM and DiI-stained MM had been ready by mixing the cell membrane with DiO or DiI on the membrane protein/dye weight ratio of 1000/1 [59].
Preparation and characterization of NCs
PG was ready in accordance with our earlier report [54]. The chemical construction and the secondary construction of PG had been decided by 1H NMR and CD, respectively. Then, PG resolution (1 mg/mL in DEPC water) was blended with siCkip-1 resolution (0.1 mg/mL in DEPC water) at numerous PG/siCkip-1 weight ratios (2.5, 5, 10, 15, 20, and 25). The combination was vortexed for five s and incubated at room temperature (RT) for 20 min to type PG/siCkip-1 (PsC) NCs. Then, CAT resolution (4 mg/mL) was added to PsC NCs at numerous CAT/siCkip-1 weight ratios (2.5, 5, 10, and 15), vortexed for five s, and incubated for 20 min to acquire the CAT-adsorbed PsC NCs (CPsC NCs). Subsequently, MM-coated CPsC NCs (M@CPsC NCs) had been fabricated utilizing the sonication technique as reported beforehand [58]. MM resolution (5 mg/mL) was added to CPsC NCs at numerous membrane protein/siCkip-1 weight ratios (5, 10, 15, and 20), adopted by sonication (2 min) to permit membrane coating. The freshly ready NCs had been subjected to electrophoresis (90 V, 20 min) in agarose gel (2%) to watch the siRNA migration. The zeta potential and hydrodynamic diameter of the freshly ready NCs had been recorded on a Zetasizer (Nano ZS 90, Malvern). The morphology of NCs was noticed by transmission electron microscopy (TEM) following adverse staining with phosphotungstic acid (1%, w/v). The steadiness of NCs in PBS (pH 7.4) containing 10% FBS was evaluated by measuring the particle dimension following incubation at RT for numerous time. To find out the membrane coating effectivity, DiD-stained MM was coated onto CPsC NCs as described above. Then, the obtained DiDM@CPsC NCs had been centrifuged (10,000 g, 10 min), and the quantity of un-coated DiD-stained MM within the supernatant was decided by spectrofluorimetry (λex = 644 nm, λem = 665 nm). The fluorescence depth of the freshly ready DiDM@CPsC NCs earlier than centrifugation was decided and set as 100%.
To organize sSDF-1α-immobilized M@CPsC NCs (DSM@CPsC NCs), DSPE-PEG2k-conjugated sSDF-1α (DS) was firstly ready by way of the press response between DSPE-PEG2k-DBCO and N3-sSDF-1α. Briefly, DSPE-PEG2k-DBCO resolution (10 mg/mL in PBS, pH = 7.4, 224 µL) was blended with N3-sSDF-1α resolution (5 mg/mL in PBS, pH = 7.4, 200 µL) and stirred at 37 °C for two h. DS was obtained after purification by ultrafiltration (MWCO = 3 kDa). The purified resolution was collected and subjected to excessive efficiency liquid chromatography (HPLC, Thermofisher) evaluation outfitted with a UV-vis detector (λabs = 214 nm) to find out the sSDF-1α focus within the remaining DS resolution. A mix of acetonitrile and water (4:1, v/v) containing 0.1% trifluoroacetic acid was used because the cell section. The freshly ready DS was blended with M@CPsC NCs at numerous membrane protein/sSDF-1α weight ratios, vortexed for five s, and incubated for 10 min to acquire DSM@CPsC NCs. The bovine serum albumin (BSA)-containing NCs (DSM@BPsC NCs) had been equally ready, whereby BSA was used as a substitute of CAT. The abbreviations of assorted NCs had been listed in Desk S3. FRET assay was performed to verify the insertion of DS into the cell membrane. Significantly, DSM@CPsC NCs had been constructed from DiO-labeled MM and Cy3-labeled DS at numerous membrane protein/sSDF-1α weight ratios. As a management, M@CPsC NCs (containing DiO-labeled MM) had been blended with Cy3-labeled N3-sSDF-1α (with out DSPE because the membrane-anchoring area) as a substitute of Cy3-labeled DS. The fluorescence emission spectrum of every pattern was recorded between 520 and 600 nm on the excitation wavelength of 480 nm. Macrophage-specific floor markers (MAC-1 and CD68) on MM and DSM@CPsC NCs had been examined by Western blot.
In vitro oxygen era and gas-driven membrane shedding
Free CAT, DSM@CPsC NCs, and DSM@BPsC NCs (0.1 mg CAT or BSA/mL) had been incubated with H2O2 (50 mM) at 37 °C for 1 h. The era of oxygen bubbles was recorded by a digital digital camera.
To watch the membrane shedding, freshly ready DSM@CPsC NCs and DSM@BPsC NCs had been handled with H2O2 (100 µM) for various time, adopted by measurement of the dimensions and zeta potential. Then, the FRET assay was additionally performed. Briefly, DSM@CPsC NCs and DSM@BPsC NCs comprised of DiI-labeled MM and FAM-siCkip-1 had been incubated with H2O2 (100 µM) for 4 h. The fluorescence emission spectra of NCs earlier than and after H2O2 therapy had been recorded between 500 and 650 nm on the excitation wavelength of 494 nm. The fluorescence restoration of the donor (FAM) at 530 nm was used to signify the membrane shedding from NCs. Lastly, confocal laser scanning microscopy (CLSM, Zeiss LSM 800) was used to watch the membrane shedding. The freshly ready DSM@CPsC NCs comprised of FAM-siCkip-1 and DiI-labeled MM had been handled with H2O2 (100 µM) for 4 h adopted by CLSM remark.
In vitro MSCs migration
The transwell tradition system was adopted to guage the sSDF-1α-mediated MSCs migration. Briefly, MSCs had been seeded onto the apical aspect of the inserts (pore dimension of 8.0 μm, Corning, NY, 1 × 106 cell/mL) and cultured for twenty-four h. Then, the cell tradition medium on the basolateral aspect was changed with recent α-MEM containing sSDF-1α, H2O2-treated (100 μm, 4 h) DSM@CPsS NCs, or untreated DSM@CPsS NCs (1 µg siScr/mL, 1 µg sSDF-1α/mL). After incubation for 48 h, the tradition medium at each the apical and basolateral sides was changed with impartial formalin (10%) and incubated for 10 min. Then, cells on the basolateral aspect of the transwell membrane had been stained with crystal violet (0.1%, 30 min), washed with PBS for 3 times, and noticed by an optical microscope. Six fields at 20× magnification had been randomly chosen to rely the variety of migrated MSCs.
Mobile uptake and intracellular distribution of NCs in MSC
MSCs had been seeded on 6-well plates (3 × 105 cells/properly) and cultured for twenty-four h. Then, numerous FAM-siCkip-1-containing NCs, together with CPsC NCs, DSM@CPsC NCs, H2O2-treated (100 µM, 4 h) DSM@CPsC NCs, and H2O2-treated (100 µM, 4 h) DSM@BPsSFAM NCs, had been added on the remaining focus of 1 µg FAM-siCkip-1/mL. After 4-h incubation, cells had been washed with PBS for 3 times, re-suspended in PBS (0.3 mL), and subjected to move cytometric (FCM, FACS Calibur, BD, USA) evaluation. Information had been analyzed utilizing the Flowjo software program.
The endo/lysosomal escape of NCs was noticed by CLSM. MSCs had been seeded on glass-bottomed dishes (2 × 104 cells/dish, 20 mm in diameter) and cultured for twenty-four h. Cells had been then incubated with H2O2-treated (100 µM, 4 h), FAM-siCkip-1-containing DSM@CPsC NCs at 1 µg FAM-siCkip-1/mL for 4 h. After washing with PBS containing sodium heparin (20 U /mL) for 3 times, cells had been stained with Lysotracker Pink (200 nM, 1.5 h) and Hoechst 33342 (10 µg/mL, 20 min) adopted by CLSM remark. The co-localization ratios had been analyzed utilizing the Picture J software program.
In vitro cytotoxicity of NCs
MSCs had been seeded on 96-well plates (8 × 103 cells/properly) and cultured for twenty-four h. Numerous NCs had been added at 1 µg siCkip-1/mL and incubated with cells for twenty-four h. The cell viability was then decided by the MTT assay. Cells handled with PBS had been used because the management to signify 100% viability.
In vitro Ckip-1 silencing and osteogenesis
MSCs had been seeded on 6-well plates (1 × 105 cells/properly) and cultured for twenty-four h. After alternative with recent medium, DSM@CPsC NCs, H2O2-treated (100 µM, 4 h) DSM@CPsS NCs, or H2O2-treated (100 µM, 4 h) DSM@CPsC NCs had been added at 1 µg siRNA/mL and incubated with cells for twenty-four h. The Ckip-1 mRNA degree in cells was decided by real-time PCR. To judge the osteogenic differentiation of MSCs, the mRNA ranges of Smad 1/5 and osteogenesis-associated genes (Runx2, Col I, ALP, and OCN) had been decided by real-time PCR. Furthermore, Ckip-1 and osteogenesis-associated proteins (Runx2, Col I, ALP, and OCN) ranges had been additionally decided by Western blot. The concentrations of major antibody and second antibody had been each 1/1000. GAPDH was used as the inner management.
To additional discover the NCs-mediated osteogenic differentiation of MSCs, MSCs had been seeded on 6-well plates (1 × 105 cells/properly) and cultured for twenty-four h. After alternative with osteo-induction medium, H2O2-treated (100 µM, 4 h) DSM@CPsC NCs or DSM@CPsS NCs had been added at 1 µg siRNA/mL. The medium containing numerous NCs was refreshed each 2 d. After 14-d incubation, cells had been washed with PBS for 3 times, fastened with impartial formalin (10%, 10 min), and stained by Alizarin purple S (ARS, 5 mg/mL, 15 min) to indicate calcium deposition. Cells had been washed with PBS for 3 times and imaged utilizing an inverted microscope (Leica TSR2). Cells had been then incubated with cetylpyridinium chloride (10%, pH = 7.0) for 15 min, and subjected to dedication of absorbance at 562 nm utilizing a microplate reader (Bio-Tek, Synergy H1).
Pharmacokinetics, biodistribution, and fracture-targeting of NCs in vivo
C57BL/6 mice had been i.v. injected with Cy5-siCkip-1-containing CPsC NCs or DSM@CPsC NCs (1 mg Cy5-siCkip-1/kg, 1 mg sSDF-1α/kg). At predetermined time factors, blood (70 µL) was collected from the orbit and blended with the passive lysis buffer (100 µL, supplemented with 1% Triton X-100). Dimethyl sulfoxide (DMSO, 200 µL) was added into the combination and incubated in a single day at RT. After centrifugation (14,800 rpm, 30 min), the focus of Cy5-siCkip-1 within the supernatant was decided by spectrofluorimetry (λex = 633 nm, λem = 678 nm).
For the analysis of the in vivo focusing on of fractured femur, femur-fractured C57BL/6 mice had been i.v. injected with Cy5-siCkip-1-containing DSM@CPsC NCs or CPsC NCs (1 mg Cy5-siCkip-1/kg, 1 mg sSDF-1α/kg) at 24 h put up fracture. At predetermined time intervals (1, 3, 6, 9, 12, and 24 h), mice had been imaged utilizing the Maestro In Vivo Imaging System. In a parallel research, mice had been sacrificed at 24 h put up injection. The key organs (coronary heart, liver, spleen, lung, and kidney) and the entire femurs had been harvested and imaged (λex = 633 nm, λem = 678 nm).
In vivo photoacoustic (PA) imaging
Femur-fractured C57BL/6 mice had been i.v. injected with PBS, DSM@BPsC NCs, or DSM@CPsC NCs (1 mg siCkip-1/kg, 1 mg sSDF-1α/kg) at 24 h put up fracture. The echo sign from O2 within the fractured femur was recorded utilizing the PA imaging system (FujiFilm VisualSonics Inc.) with the PA mode (Oxy-hem mode, 750 and 850 nm) at 3 h put up injection.
In vivo membrane shedding
The in vivo membrane shedding from NCs was monitored by the FRET assay. Femur-fractured C57BL/6 mice had been i.v. injected with DSM@CPsC NCs or DSM@BPsC NCs comprised of Cy5-siCkip-1 and DiI-labeled MM (1 mg Cy5-siCkip-1/kg). At 6 h put up injection, mice had been sacrificed, and the fractured femurs had been harvested and imaged utilizing the Maestro In Vivo Imaging System (λex = 550 nm, λem = 670 nm). The disappearance of the acceptor (Cy5) sign was used to signify the separation of MM and siCkip-1.
In vivo uptake of NCs by MSCs
Femur-fractured C57BL/6 mice had been i.v. injected with FAM-siCkip-1-containing NCs (DSM@CPsC NCs and DSM@BPsC NCs) at 1 mg FAM-siCkip-1/kg. At 24 h put up injection, mice had been sacrificed, and the fractured femurs had been harvested, flushed with sterile PBS, grinded with a mortar, and digested with enzymes (2 mg/mL collagenase, 100 µg/mL DNase, and 20 µg/mL RNase) for 1 h to arrange mono-dispersed cell suspensions. Cells had been collected by way of centrifugation (500 g, 3 min), stained with antibodies (PerCP/Cy5.5-labeled anti-CD45, PE-labeled anti-CD73, and APC-labeled anti-CD105) for 0.5 h, washed with PBS for 3 times, resuspended within the FACS buffer (PBS containing 1% FBS, 0.2 mL), and subjected to FCM evaluation. Cells had been first gated utilizing FSC/SSC, adopted by CD45, CD73, and CD105 gating to determine CD45−CD73+CD105+ populations (MSCs) for the dedication of MSCs that had taken up FAM-siCkip-1-containing NCs.
In vivo MSCs recruitment, Ckip-1 silencing, and osteogenesis
Femur-fractured C57BL/6 mice had been i.v. injected with PBS, DSM@CPsS NCs, DSM@CPsC NCs, or M@CPsC NCs (1 mg siRNA/kg, 1 mg sSDF-1α/kg) on day 1, 3, and 5 put up fracture. 5 millimeter-length bones spanning the fracture websites had been harvested on day 7, flushed with sterile PBS, grinded with a mortar, and digested with enzymes (2 mg/mL collagenase, 100 µg/mL DNase, and 20 µg/mL RNase) for 1 h to arrange mono-dispersed cell suspensions. Cells had been then washed with PBS, stained with antibodies (PerCP/Cy5.5-labeled anti-CD45, PE-labeled anti-CD73, and APC-labeled anti-CD105), washed with PBS, and subjected to FCM evaluation. In a parallel research, femurs had been harvested on day 7 or 28 put up fracture. The mRNA ranges of Ckip-1 and osteogenesis-associated genes (Runx2, Col I, ALP, and OCN) in femurs on day 7 had been decided by real-time PCR following reported protocols [59]. The Ckip-1 protein degree on day 7 was additional decided by Western blot as described earlier than [54]. The expression ranges of osteogenesis-associated proteins (Runx2, Col I, ALP, OCN, and OPG) on day 28 had been decided by immunofluorescence (IFC) or immunohistochemical (IHC) staining and quantified utilizing the ImageJ software program. Information had been calculated because the imply fluorescence depth (for IFC) or imply grey worth (for IHC) of the optimistic cells.
X-ray and micro-CT imaging
Femur-fractured C57BL/6 mice had been i.v. injected with DSM@CPsS NCs, DSM@CPsC NCs, or M@CPsC NCs (1 mg siRNA/kg, 1 mg sSDF-1α/kg) on day 1, 3, and 5 put up fracture. Mice had been imaged with an X-ray imaging system (Faxitron MX-20, Tucson, AZ) on day 14 and 28. The radiographs on day 28 had been scored for callus opacity, cortical transforming/bridging, and periosteal/endosteal response by three impartial assessors blinded to grouping and complete scores had been calculated [63]. Callus opacity and periosteal/endosteal response had been scored by the obvious radiographic density and the importance degree of periosteal/endosteal response, respectively. Scoring vary for each callus opacity and periosteal/endosteal response went from 0 (none) to three (marked). Cortical transforming/bridging was scored by visibility and mineralization of cortical edges, and scores ranged from 0 (none) to 4 (full union with properly demarcated medullary canal).
In a parallel research, femurs had been harvested on day 28 put up fracture and scanned utilizing micro-CT (Skyscan 1176, Belgium) as described beforehand [62]. Excessive decision scanograms (9–20 mm) had been obtained (decision: 8.8 mm, supply voltage: 50 kV, supply present: 500 mA, rotation step: 0.7 unit). The info set was reconstructed utilizing the CT analyzer software program (Skyscan) to acquire the 3D pictures of femur and to measure morphometric parameters. The area of curiosity (ROI) within the fractured femur was chosen for the dedication of bone mineral density (BMD), trabecular quantity (Tb.N), and trabecular separation/spacing (Tb.Sp).
Histological evaluation
Femurs had been harvested on day 28 put up fracture as described above. Extra tender tissues and pores and skin had been eliminated. Femurs had been fastened in 4% impartial formalin for 3 d, decalcified in 10% ethylenediaminetetraacetic acid resolution for one month at RT, embedded in paraffin, and sliced at 6 μm in thickness. The femur sections had been stained with Sirius purple, Masson’s trichrome (MT), haematoxylin and eosin (H&E), or Safranin O/quick inexperienced to guage the Col I expression, complete collagen deposition, new bone formation, and bone mineralization, respectively, as vital indexes for osteogenesis. The contents of Col I (vibrant purple/yellow collagen fiber), deposited complete collagen (darkish blue), newly shaped bone (together with lamellar bone and cartilage), and mineralized bone (inexperienced space) had been decided utilizing the ImageJ software program. Information had been denoted as the share of stain-positive space to the full space in every part.
Biosafety evaluation of DSM@CPsC NCs
Wholesome C57BL/6 mice had been i.v. injected with PBS or DSM@CPsC NCs (1 mg siCkip-1/kg) for 3 times with 24 h spaced between every injection. Blood was collected at 24 h put up the final injection adopted by hematological and biochemical analyses. Main organs had been additionally harvested and subjected to histological evaluation utilizing H&E staining.
Statistical evaluation
All information had been introduced as means ± customary deviations, and statistical evaluation was carried out utilizing One-way ANOVA. The variations between two experimental teams had been judged to be vital at *p < 0.05 and really vital at **p < 0.01 and ***p < 0.001.
