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 Expertise Co., LTD. (Xi’an, China). α-Helical polypeptide bearing guanidine teams on the aspect chains (PG, polymerization diploma = 102) was synthesized in response to our earlier report [54]. Lysotracker Crimson and Trizol reagent have been bought from Thermo Fisher Scientific (Massachusetts, USA). BCA protein assay equipment, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), and 4’,6-diamidino-2-phenylindole (DAPI) have been bought from Alfa Aesar (Shanghai, China). PrimerScript real-time reagent equipment and SYBR Premix Ex Taq equipment have been bought from Takara Bio (Qingdao, China). Azide-modified sSDF-1α peptide (N3-KSKPVVLSYR, N3-sSDF-1α) and Cy3-labeled N3-sSDF-1α have been bought from Bankpeptide Biotechnology Co., LTD. (Hefei, China). Major antibodies (anti-Col I, anti-ALP, anti-Runx2, anti-OCN, and anti-OPG) have 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 have been bought from Absin Bioscience Co., LTD. (Shanghai, China). APC-labeled anti-CD105, PE-labeled anti-CD73, and Percy/Cy5.5-labeled anti-CD45 have been bought from Elabscience Biotechnology Co., LTD (Wuhan, China). siCkip-1 and damaging management siRNA with scrambled sequence (siScr) have been bought from GenePharma Co., LTD. (Shanghai, China), and their sequences have been listed in Supplementary: Desk S1. All primers have been bought from Sangong Biotech (Shanghai, China), and their sequences have been listed in Supplementary: Desk S2. All the opposite reagents have been bought from Power Chemical (Shanghai, China) or Aladdin (Shanghai, China) and used as obtained.
Animals and cells
Feminine C57BL/6 mice (6–8 weeks, 18–20 g) have been bought from Slaccas Experimental Animal Co., LTD. (Shanghai, China) and housed in a particular pathogen-free (SPF) animal lab.
RAW 264.7 cells (mouse monocyte macrophage) have been bought from the American Sort Tradition Assortment (Rockville, MD, USA) and cultured in DMEM containing 10% fetal bovine serum (FBS). Mouse MSCs (bone marrow mesenchymal stem cells) have 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 response to a earlier report [43]. Significantly, mice have been anesthetized, and their proper hips, thighs, and knees have 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 correct femur of every mouse utilizing a rotary Dremel with a diamond blade attachment. The patella was then lowered, and the incision was closed utilizing 4 − 0 artificial suture. Mice have been allowed to maneuver freely after restoration from anesthesia. On day 5 publish fracture, animals have 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 response to a earlier report [62]. Briefly, RAW 264.7 cells on the tradition dish (60 mm in diameter) have 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 equipment. For fluorescence microscopy imaging and fluorescence resonance power switch (FRET) evaluation, DiO-stained MM and DiI-stained MM have 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 response to our earlier report [54]. The chemical construction and the secondary construction of PG have 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 kind 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) have been fabricated utilizing the sonication methodology 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 have 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 have been recorded on a Zetasizer (Nano ZS 90, Malvern). The morphology of NCs was noticed by transmission electron microscopy (TEM) following damaging staining with phosphotungstic acid (1%, w/v). The soundness of NCs in PBS (pH 7.4) containing 10% FBS was evaluated by measuring the particle measurement 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 have 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 through the clicking 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 geared up with a UV-vis detector (λabs = 214 nm) to find out the sSDF-1α focus within the closing DS resolution. A mix of acetonitrile and water (4:1, v/v) containing 0.1% trifluoroacetic acid was used because the cell part. 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) have been equally ready, whereby BSA was used as a substitute of CAT. The abbreviations of varied NCs have been listed in Desk S3. FRET assay was carried out to verify the insertion of DS into the cell membrane. Significantly, DSM@CPsC NCs have 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) have 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 have been examined by Western blot.
In vitro oxygen technology and gas-driven membrane shedding
Free CAT, DSM@CPsC NCs, and DSM@BPsC NCs (0.1 mg CAT or BSA/mL) have been incubated with H2O2 (50 mM) at 37 °C for 1 h. The technology of oxygen bubbles was recorded by a digital digicam.
To watch the membrane shedding, freshly ready DSM@CPsC NCs and DSM@BPsC NCs have been handled with H2O2 (100 µM) for various time, adopted by measurement of the scale and zeta potential. Then, the FRET assay was additionally carried out. Briefly, DSM@CPsC NCs and DSM@BPsC NCs comprised of DiI-labeled MM and FAM-siCkip-1 have been incubated with H2O2 (100 µM) for 4 h. The fluorescence emission spectra of NCs earlier than and after H2O2 therapy have 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 have been handled with H2O2 (100 µM) for 4 h adopted by CLSM statement.
In vitro MSCs migration
The transwell tradition system was adopted to judge the sSDF-1α-mediated MSCs migration. Briefly, MSCs have been seeded onto the apical aspect of the inserts (pore measurement 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 contemporary 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 have been stained with crystal violet (0.1%, 30 min), washed with PBS for thrice, and noticed by an optical microscope. Six fields at 20× magnification have been randomly chosen to rely the variety of migrated MSCs.
Mobile uptake and intracellular distribution of NCs in MSC
MSCs have been seeded on 6-well plates (3 × 105 cells/nicely) 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, have been added on the closing focus of 1 µg FAM-siCkip-1/mL. After 4-h incubation, cells have been washed with PBS for thrice, re-suspended in PBS (0.3 mL), and subjected to circulate cytometric (FCM, FACS Calibur, BD, USA) evaluation. Knowledge have been analyzed utilizing the Flowjo software program.
The endo/lysosomal escape of NCs was noticed by CLSM. MSCs have been seeded on glass-bottomed dishes (2 × 104 cells/dish, 20 mm in diameter) and cultured for twenty-four h. Cells have 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 thrice, cells have been stained with Lysotracker Crimson (200 nM, 1.5 h) and Hoechst 33342 (10 µg/mL, 20 min) adopted by CLSM statement. The co-localization ratios have been analyzed utilizing the Picture J software program.
In vitro cytotoxicity of NCs
MSCs have been seeded on 96-well plates (8 × 103 cells/nicely) and cultured for twenty-four h. Varied NCs have 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 have been used because the management to signify 100% viability.
In vitro Ckip-1 silencing and osteogenesis
MSCs have been seeded on 6-well plates (1 × 105 cells/nicely) and cultured for twenty-four h. After substitute with contemporary medium, DSM@CPsC NCs, H2O2-treated (100 µM, 4 h) DSM@CPsS NCs, or H2O2-treated (100 µM, 4 h) DSM@CPsC NCs have 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 guage the osteogenic differentiation of MSCs, the mRNA ranges of Smad 1/5 and osteogenesis-associated genes (Runx2, Col I, ALP, and OCN) have been decided by real-time PCR. Furthermore, Ckip-1 and osteogenesis-associated proteins (Runx2, Col I, ALP, and OCN) ranges have been additionally decided by Western blot. The concentrations of main antibody and second antibody have been each 1/1000. GAPDH was used as the interior management.
To additional discover the NCs-mediated osteogenic differentiation of MSCs, MSCs have been seeded on 6-well plates (1 × 105 cells/nicely) and cultured for twenty-four h. After substitute with osteo-induction medium, H2O2-treated (100 µM, 4 h) DSM@CPsC NCs or DSM@CPsS NCs have been added at 1 µg siRNA/mL. The medium containing numerous NCs was refreshed each 2 d. After 14-d incubation, cells have been washed with PBS for thrice, mounted with impartial formalin (10%, 10 min), and stained by Alizarin purple S (ARS, 5 mg/mL, 15 min) to indicate calcium deposition. Cells have been washed with PBS for thrice and imaged utilizing an inverted microscope (Leica TSR2). Cells have 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 have 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 have 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 publish fracture. At predetermined time intervals (1, 3, 6, 9, 12, and 24 h), mice have been imaged utilizing the Maestro In Vivo Imaging System. In a parallel examine, mice have been sacrificed at 24 h publish injection. The main organs (coronary heart, liver, spleen, lung, and kidney) and the entire femurs have been harvested and imaged (λex = 633 nm, λem = 678 nm).
In vivo photoacoustic (PA) imaging
Femur-fractured C57BL/6 mice have 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 publish 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 publish injection.
In vivo membrane shedding
The in vivo membrane shedding from NCs was monitored by the FRET assay. Femur-fractured C57BL/6 mice have 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 publish injection, mice have been sacrificed, and the fractured femurs have 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 have 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 publish injection, mice have been sacrificed, and the fractured femurs have 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 have been collected through 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 thrice, resuspended within the FACS buffer (PBS containing 1% FBS, 0.2 mL), and subjected to FCM evaluation. Cells have 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 have 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 publish fracture. 5 millimeter-length bones spanning the fracture websites have 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 have 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 examine, femurs have been harvested on day 7 or 28 publish fracture. The mRNA ranges of Ckip-1 and osteogenesis-associated genes (Runx2, Col I, ALP, and OCN) in femurs on day 7 have 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 have been decided by immunofluorescence (IFC) or immunohistochemical (IHC) staining and quantified utilizing the ImageJ software program. Knowledge have been calculated because the imply fluorescence depth (for IFC) or imply grey worth (for IHC) of the constructive cells.
X-ray and micro-CT imaging
Femur-fractured C57BL/6 mice have 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 publish fracture. Mice have been imaged with an X-ray imaging system (Faxitron MX-20, Tucson, AZ) on day 14 and 28. The radiographs on day 28 have been scored for callus opacity, cortical transforming/bridging, and periosteal/endosteal response by three impartial assessors blinded to grouping and complete scores have been calculated [63]. Callus opacity and periosteal/endosteal response have 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 nicely demarcated medullary canal).
In a parallel examine, femurs have been harvested on day 28 publish fracture and scanned utilizing micro-CT (Skyscan 1176, Belgium) as described beforehand [62]. Excessive decision scanograms (9–20 mm) have 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 photos 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 have been harvested on day 28 publish fracture as described above. Extra mushy tissues and pores and skin have been eliminated. Femurs have been mounted 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 have been stained with Sirius purple, Masson’s trichrome (MT), haematoxylin and eosin (H&E), or Safranin O/quick inexperienced to judge the Col I expression, complete collagen deposition, new bone formation, and bone mineralization, respectively, as necessary indexes for osteogenesis. The contents of Col I (vivid purple/yellow collagen fiber), deposited complete collagen (darkish blue), newly fashioned bone (together with lamellar bone and cartilage), and mineralized bone (inexperienced space) have been decided utilizing the ImageJ software program. Knowledge have been denoted as the proportion of stain-positive space to the overall space in every part.
Biosafety evaluation of DSM@CPsC NCs
Wholesome C57BL/6 mice have been i.v. injected with PBS or DSM@CPsC NCs (1 mg siCkip-1/kg) for thrice with 24 h spaced between every injection. Blood was collected at 24 h publish the final injection adopted by hematological and biochemical analyses. Main organs have been additionally harvested and subjected to histological evaluation utilizing H&E staining.
Statistical evaluation
All information have been introduced as means ± commonplace deviations, and statistical evaluation was carried out utilizing One-way ANOVA. The variations between two experimental teams have been judged to be vital at *p < 0.05 and really vital at **p < 0.01 and ***p < 0.001.
