An anti-inflammatory peptide and brain-derived neurotrophic factor-modified hyaluronan-methylcellulose hydrogel promotes nerve regeneration in rats with spinal cord injury methods
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Methods
The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation.
Materials and methods
Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dialysis, sulfhydryl groups (100 mg of reactive thiols) were integrated into the carboxylated MC polymer (250 mg) main chain upon reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (750 mg) and 3,3′-dithiobis (propionic dihydrazide, 750 mg), followed by reduction of the disulfide bond with dithiothreitol (1.0 g). Maleimide-labeled streptavidin (mol maleimide: mol streptavidin =9:1, 1.1 mg) and maleimide-KAFAK peptide (59 mg) were then added and mixed with previously synthesized sulfhydryl-MC by stirring in 0.1 M PBS (100 mL) overnight at 4&#...
Materials and methods
HAMC-KAFAK/BDNF hydrogels were prepared by physical blending of HA, MC-KAFAK, MC-streptavidin, and biotinylated-BDNF in artificial cerebrospinal fluid (aCSF) with a final composition of 1.5 wt% HA, 2.5 wt% MC-KAFAK, 0.5 wt% MC-streptavidin, and 0.2 wt% biotinylated BDNF. The HAMC hydrogels were prepared with 1.5 wt% HA and 3 wt% MC for comparison. The above ingredients were sterile-filtered before mixing and sequentially added to the aCSF using a high-speed centrifugal mixer (Eppendorf) for 25 seconds at 3,000 rpm/min and dissolved at 4°C for 12 hours. For in vivo studies, the HAMC-KAFAK/BDNF hydrogel compositions were sterilized by filtration and the preparation process was in sterile conditions. The time interval between preparation and use of the final composite hydrogels was less than 15 minutes, during which time the hydrogels were kept at 4°C ( ).
Swelling properties of HAMC-KAFAK/BDNF hydrogels
Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o × 100%. W t is the weight of the swollen hydrogel and W o is the initial weight. The SR was calculated at 6, 12, 18, and 24 hours.
Cell culture and cell proliferation assay
PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated at 37°C, 5% CO 2, and 95% humidity. The cultivated medium was replaced every 2 days. Cells were passaged when confluence reached 80%-90%. HAMC-KAFAK/BDNF treatment was used to evaluate the effect of released BDNF on PC12 cells proliferation. The PC12 cells were seeded at the density of 1 × 10 4 cells/cm 2 into 96-well plates and 100 µL medium per well. Cells were treated with HAMC-KAFAK/BDNF hydrogels, while the same volume of DMEM was added to the control group. The PC12 cells proliferation was evaluated with a CCK-8 cell proliferation kit (Dojindo Molec...
KAFAK and BDNF release in vitro
To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis. Fluorescence measurements of KAFAK release were taken at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed.
KAFAK and BDNF release in vitro
To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed. Finally, the accumulated release ratio was calculated.
Animals and surgical procedures
All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from the Laboratory Animal Center of the Military Medical Science Academy of China were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. All 72 rats were randomized to 4 groups: sham, SCI, HAMC, and HAMC-KAFAK/BDNF (n=18). A laminectomy was performed at the T10 vertebral level to expose the spinal cord and an aneurysm clip with a force of 25 × g was used for 1 minute to develop an SCI model. A HAMC-KAFAK/BDNF or HAMC hydrogel injection was administered after 5 minutes of SCI. A Hamilton syringe was inserted into th...
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The anti-inflammatory peptide KAFAKLAARLYRKALARQLGVAA (KAFAK) and brain-derived neurotrophic factor (BDNF)-modified hyaluronan-methylcellulose (HAMC) hydrogel was designed for minimally invasive, localized, and sustained intrathecal protein delivery. The physical and biological characteristics of HAMC-KAFAK/BDNF hydrogel were measured in vitro. SCI model was performed in rats and HAMC-KAFAK/BDNF hydrogel was injected into the injured site of spinal cord. The neuronal regeneration effect was evaluated by inflammatory cytokine levels, behavioral test and histological analysis at 8 weeks post operation.
Maleimide-KAFAK and maleimide-streptavidin were covalently bonded to the MC by thiol-maleimide click chemistry and biotinylated BDNF was conjugated to MC-streptavidin as previously described. Briefly, MC is carboxylated using 1.5 M sodium hydroxide and an overdose of bromoacetic acid. After purification by dialysis, sulfhydryl groups (100 mg of reactive thiols) were integrated into the carboxylated MC polymer (250 mg) main chain upon reaction with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (750 mg) and 3,3′-dithiobis (propionic dihydrazide, 750 mg), followed by reduction of the disulfide bond with dithiothreitol (1.0 g). Maleimide-labeled streptavidin (mol maleimide: mol streptavidin =9:1, 1.1 mg) and maleimide-KAFAK peptide (59 mg) were then added and mixed with previously synthesized sulfhydryl-MC by stirring in 0.1 M PBS (100 mL) overnight at 4°C. N-ethylhydroxy maleimide (13 mg) was then added to quench unreacted free sulfhydryl groups, followed by the removal of unbound maleimide-containing reagent by dialysis. Then MC-KAFAK and MC-streptavidin were sterile-filtered using a 0.22 µm syringe filter and then lyophilized to amorphou...
HAMC-KAFAK/BDNF hydrogels were prepared by physical blending of HA, MC-KAFAK, MC-streptavidin, and biotinylated-BDNF in artificial cerebrospinal fluid (aCSF) with a final composition of 1.5 wt% HA, 2.5 wt% MC-KAFAK, 0.5 wt% MC-streptavidin, and 0.2 wt% biotinylated BDNF. The HAMC hydrogels were prepared with 1.5 wt% HA and 3 wt% MC for comparison. The above ingredients were sterile-filtered before mixing and sequentially added to the aCSF using a high-speed centrifugal mixer (Eppendorf) for 25 seconds at 3,000 rpm/min and dissolved at 4°C for 12 hours. For in vivo studies, the HAMC-KAFAK/BDNF hydrogel compositions were sterilized by filtration and the preparation process was in sterile conditions. The time interval between preparation and use of the final composite hydrogels was less than 15 minutes, during which time the hydrogels were kept at 4°C ( ).
Swelling properties were measured in physiologic conditions. Hydrogels were added into 50 mL centrifuge tubes with PBS at 37°C. At different time points, the hydrogels were taken out, and the weight was measured after removal of excess water on the surface. The swelling ratio (SR) = ( W t - W o )/ W o × 100%. W t is the weight of the swollen hydrogel and W o is the initial weight. The SR was calculated at 6, 12, 18, and 24 hours.
PC12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were cultured in DMEM (Gibco ®; Thermo Fisher Scientific, Waltham, MA, USA) with 10% fetal bovine serum (Thermo Fisher Scientific), 0.1 mg/mL streptomycin, and 100 U/mL penicillin. Cultures were incubated at 37°C, 5% CO 2, and 95% humidity. The cultivated medium was replaced every 2 days. Cells were passaged when confluence reached 80%-90%. HAMC-KAFAK/BDNF treatment was used to evaluate the effect of released BDNF on PC12 cells proliferation. The PC12 cells were seeded at the density of 1 × 10 4 cells/cm 2 into 96-well plates and 100 µL medium per well. Cells were treated with HAMC-KAFAK/BDNF hydrogels, while the same volume of DMEM was added to the control group. The PC12 cells proliferation was evaluated with a CCK-8 cell proliferation kit (Dojindo Molecular Technologies, Inc., Japan). In brief, at 1, 3, and 7 days, 10 µL CCK-8 solution was applied to each well and incubated for 1 hour, and the absorbance at 450 nm was measured. The experiments were performed in triplicate.
To explore the profile of KAFAK release by the HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of KAFAK: 0.6 µg/mL) to 2 mL sterile PBS. KAFAK release into solution was measured using a fluoraldehyde o-phthalaldehyde assay (Thermo Fisher Scientific), by fluorescence analysis. Fluorescence measurements of KAFAK release were taken at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed.
To explore the profile of BDNF release by HAMC hydrogel, we added 25 µL of HAMC-KAFAK/BDNF hydrogels (the concentration of BDNF: 1 µg/mL) to 2 mL sterile PBS. BDNF release into solution was measured using a rat BDNF ELISA kit (Beijing Solarbio Science & Technology Co., Ltd). Supernatant was collected at 6, 12, 24, 48, 72, and 96 hours. Three replicates were performed. Finally, the accumulated release ratio was calculated.
All procedures were conducted according to protocols approved by Institutional Animal Care and Use Committee of Logistics College of PAP and all experiments were approved by the Institutional Animal Care and Use Committee of Logistics College of PAP. Adult female Sprague Dawley rats (200-230 g) obtained from the Laboratory Animal Center of the Military Medical Science Academy of China were anesthetized with a nitrous oxide/oxygen mixture (70%/30%) containing 1.4% isoflurane delivered by nose cone. All 72 rats were randomized to 4 groups: sham, SCI, HAMC, and HAMC-KAFAK/BDNF (n=18). A laminectomy was performed at the T10 vertebral level to expose the spinal cord and an aneurysm clip with a force of 25 × g was used for 1 minute to develop an SCI model. A HAMC-KAFAK/BDNF or HAMC hydrogel injection was administered after 5 minutes of SCI. A Hamilton syringe was inserted into the center of the traumatic area and 10 µL of the HAMC-KAFAK/BDNF or HAMC hydrogel was injected manually. Following implantation, the muscle and skin of the surgical wound were closed. Animals in the sham group received only the laminectomy without the SCI. The rats were then placed on warming pa...
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