The fabrication and related properties study of chitosan-poly (lactide-co-glycolide) double-walled microspheres loaded with nerve growth factor_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

RONG Mengyao ¹ , CHANG Zhen ² , OU Jiawei ² , ZHAO Songchuan ² , ZENG Wen ² ,  LIU Qi ³

1. Department of Internal Medicine, the Hospital of Xidian University, Xi’an Shaanxi, 710071, P.R.China;
2. Department of Spinal Surgery, Honghui Hospital Affiliated to Medical School of Xi’an Jiaotong University, Xi’an Shaanxi, 710054, P.R.China;
3. Department of Neurosurgery, the First Hospital of Yulin, Yulin Shaanxi, 718000, P.R.China;

Corresponding author：

LIU Qi, Email: xbmdlq@126.com

Keywords：

Chitosan; poly (lactide-co-glycolide); microspheres; nerve growth factor; nerve injury

DOI：

10.7507/1002-1892.201905074

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Objective To evaluate the feasibility of the chitosan-poly (lactide-co-glycolide) (PLGA) double-walled microspheres for sustained release of bioactive nerve growth factor (NGF) in vitro.Methods NGF loaded chitosan-PLGA double-walled microspheres were prepared by emulsion-ionic method with sodium tripolyphosphate (TPP) as an ionic cross-linker. The double-walled microspheres were cross-linked by different concentrations of TPP [1%, 3%, 10% (W/V)]. NGF loaded PLGA microspheres were also prepared. The outer and inner structures of double-walled microspheres were observed by light microscopy, scanning electron microscopy, confocal laser scanning microscopy, respectively. The size and distribution of microspheres and fourier transform infra red spectroscopy (FT-IR) were analyzed. PLGA microspheres with NGF or chitosan-PLGA double-walled microspheres cross-linked by 1%, 3%, and 10%TPP concentration (set as groups A, B, C, and D respectively) were used to determine the degradation ratio of microspheres in vitro and the sustained release ratio of NGF in microspheres at different time points. The bioactivity of NGF (expressed as the percentage of PC12 cells with positive axonal elongation reaction) in the sustained release solution of chitosan-PLGA double-walled microspheres without NGF (set as group A1) was compared in groups B, C, and D.Results The chitosan-PLGA double-walled microspheres showed relative rough and spherical surfaces without aggregation. Confocal laser scanning microscopy showed PLGA microspheres were evenly uniformly distributed in the chitosan-PLGA double-walled microspheres. The particle size of microspheres ranged from 18.5 to 42.7 μm. The results of FT-IR analysis showed ionic interaction between amino groups and phosphoric groups of chitosan in double-walled microspheres and TPP. In vitro degradation ratio analysis showed that the degradation ratio of double-walled microspheres in groups B, C, and D appeared faster in contrast to that in group A. In addition, the degradation ratio of double-walled microsphere in groups B, C, and D decreased when the TPP concentration increased. There were significant differences in the degradation ratio of each group (P<0.05). In vitro sustained release ratio of NGF showed that when compared with PLGA microspheres in group A, double-walled microspheres in groups B, C, and D released NGF at a relatively slow rate, and the sustained release ratio decreased with the increase of TPP concentration. Except for 84 days, there was significant difference in the sustained release ratio of NGF between groups B, C, and D (P<0.05). The bioactivity of NGF results showed that the percentage of PC12 cells with positive axonal elongation reaction in groups B, C, and D was significantly higher than that in group A1 (P<0.05). At 7 and 28 days of culture, there was no significant difference between groups B, C, and D (P>0.05); at 56 and 84 days of culture, the percentage of PC12 cells with positive axonal elongation reaction in groups C and D was significantly higher than that in group B (P<0.05), and there was no significant difference between groups C and D (P>0.05).Conclusion NGF loaded chitosan-PLGA double-walled microspheres have a potential clinical application in peripheral nerve regeneration after injury.

Citation： RONG Mengyao, CHANG Zhen, OU Jiawei, ZHAO Songchuan, ZENG Wen, LIU Qi. The fabrication and related properties study of chitosan-poly (lactide-co-glycolide) double-walled microspheres loaded with nerve growth factor. Chinese Journal of Reparative and Reconstructive Surgery, 2020, 34(1): 102-108. doi: 10.7507/1002-1892.201905074 Copy

1.	Koulaxouzidis G, Reutter W, Hildebrandt H, et al. In vivo stimulation of early peripheral axon regeneration by N-propionylmannosamine in the presence of polysialyltransferase ST8SIA2. J Neural Transm (Vienna), 2015, 122(9): 1211-1219.
2.	Oh SH, Kang JG, Kim TH, et al. Enhanced peripheral nerve regeneration through asymmetrically porous nerve guide conduit with nerve growth factor gradient. J Biomed Mater Res A, 2018, 106(1): 52-64.
3.	Umeda K, Negishi M, Katoh H. RasGRF1 mediates brain-derived neurotrophic factor-induced axonal growth in primary cultured cortical neurons. Biochem Biophys Rep, 2018, 17: 56-64.
4.	Kao HT, Ryoo K, Lin A, et al. Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts. Eur J Neurosci, 2017, 45(8): 1085-1101.
5.	Whitehead TJ, Avila COC, Sundararaghavan HG. Combining growth factor releasing microspheres within aligned nanofibers enhances neurite outgrowth. J Biomed Mater Res A, 2018, 106(1): 17-25.
6.	Hu LN, Tian JX, Gao W, et al. Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion. Neural Regen Res, 2018, 13(3): 477-483.
7.	Yao Y, Cui Y, Zhao Y, et al. Effect of longitudinally oriented collagen conduit combined with nerve growth factor on nerve regeneration after dog sciatic nerve injury. J Biomed Mater Res B Appl Biomater, 2018, 106(6): 2131-2139.
8.	Song Z, Wang Z, Shen J, et al. Nerve growth factor delivery by ultrasound-mediated nanobubble destruction as a treatment for acute spinal cord injury in rats. Int J Nanomedicine, 2017, 12: 1717-1729.
9.	Zhai P, Chen XB, Schreyer DJ. PLGA/alginate composite microspheres for hydrophilic protein delivery. Mater Sci Eng C Mater Biol Appl, 2015, 56: 251-259.
10.	Niu X, Feng Q, Wang M, et al. In vitro degradation and release behavior of porous poly (lactic acid) scaffolds containing chitosan microspheres as a carrier for BMP-2-derived synthetic peptide. Polymer Degradation and Stability, 2009, 94(2): 176-182.
11.	Yang Y, Liu M, Gu Y, et al. Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells. Cell Biol Int, 2009, 33(3): 352-356.
12.	陈伟, 刘仲前, 岳元辉, 等. bFGF 壳聚糖微球的制备及检测. 中国修复重建外科杂志, 2012, 26(8): 989-992.
13.	Zeng W, Liu Z, Li Y, et al. Development and characterization of cores-shell poly (lactide-co-glycolide)-chitosan microparticles for sustained release of GDNF. Colloids Surf B Biointerfaces, 2017, 159: 791-799.
14.	Zhang W, Gao Y, Zhou Y, et al. Localized and sustained delivery of erythropoietin from PLGA microspheres promotes functional recovery and nerve regeneration in peripheral nerve injury. Biomed Res Int, 2015, 2015: 478103.
15.	Ding T, Zhu C, Yin JB, et al. Slow-releasing rapamycin-coated bionic peripheral nerve scaffold promotes the regeneration of rat sciatic nerve after injury. Life Sci, 2015, 122: 92-99.
16.	Xu X, Qiu S, Zhang Y, et al. PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation. Artif Cells Nanomed Biotechnol, 2017, 45(2): 330-339.
17.	Li G, Zhao X, Zhang L, et al. Regulating Schwann cells growth by chitosan micropatterning for peripheral nerve regeneration in vitro. Macromol Biosci, 2014, 14(8): 1067-1075.
18.	Liu H, Wen W, Hu M, et al. Chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects. Neural Regen Res, 2013, 8(33): 3139-3147.
19.	Wang M, Feng Q, Niu X, et al. A spheres-in-sphere structure for improving protein-loading poly (lactide-co-glycolide) microspheres. Polymer Degradation and Stability, 2010, 95(1): 6-13.
20.	Tao C, Huang J, Lu Y, et al. Development and characterization of GRGDSPC-modified poly(lactide-co-glycolide acid) porous microspheres incorporated with protein-loaded chitosan microspheres for bone tissue engineering. Colloids Surf B Biointerfaces, 2014, 122: 439-446.
21.	Chen MM, Cao H, Liu YY, et al. Sequential delivery of chlorhexidine acetate and bFGF from PLGA-glycol chitosan core-shell microspheres. Colloids Surf B Biointerfaces, 2017, 151: 189-195.

1. Koulaxouzidis G, Reutter W, Hildebrandt H, et al. In vivo stimulation of early peripheral axon regeneration by N-propionylmannosamine in the presence of polysialyltransferase ST8SIA2. J Neural Transm (Vienna), 2015, 122(9): 1211-1219.
2. Oh SH, Kang JG, Kim TH, et al. Enhanced peripheral nerve regeneration through asymmetrically porous nerve guide conduit with nerve growth factor gradient. J Biomed Mater Res A, 2018, 106(1): 52-64.
3. Umeda K, Negishi M, Katoh H. RasGRF1 mediates brain-derived neurotrophic factor-induced axonal growth in primary cultured cortical neurons. Biochem Biophys Rep, 2018, 17: 56-64.
4. Kao HT, Ryoo K, Lin A, et al. Synapsins regulate brain-derived neurotrophic factor-mediated synaptic potentiation and axon elongation by acting on membrane rafts. Eur J Neurosci, 2017, 45(8): 1085-1101.
5. Whitehead TJ, Avila COC, Sundararaghavan HG. Combining growth factor releasing microspheres within aligned nanofibers enhances neurite outgrowth. J Biomed Mater Res A, 2018, 106(1): 17-25.
6. Hu LN, Tian JX, Gao W, et al. Electroacupuncture and moxibustion promote regeneration of injured sciatic nerve through Schwann cell proliferation and nerve growth factor secretion. Neural Regen Res, 2018, 13(3): 477-483.
7. Yao Y, Cui Y, Zhao Y, et al. Effect of longitudinally oriented collagen conduit combined with nerve growth factor on nerve regeneration after dog sciatic nerve injury. J Biomed Mater Res B Appl Biomater, 2018, 106(6): 2131-2139.
8. Song Z, Wang Z, Shen J, et al. Nerve growth factor delivery by ultrasound-mediated nanobubble destruction as a treatment for acute spinal cord injury in rats. Int J Nanomedicine, 2017, 12: 1717-1729.
9. Zhai P, Chen XB, Schreyer DJ. PLGA/alginate composite microspheres for hydrophilic protein delivery. Mater Sci Eng C Mater Biol Appl, 2015, 56: 251-259.
10. Niu X, Feng Q, Wang M, et al. In vitro degradation and release behavior of porous poly (lactic acid) scaffolds containing chitosan microspheres as a carrier for BMP-2-derived synthetic peptide. Polymer Degradation and Stability, 2009, 94(2): 176-182.
11. Yang Y, Liu M, Gu Y, et al. Effect of chitooligosaccharide on neuronal differentiation of PC-12 cells. Cell Biol Int, 2009, 33(3): 352-356.
12. 陈伟, 刘仲前, 岳元辉, 等. bFGF 壳聚糖微球的制备及检测. 中国修复重建外科杂志, 2012, 26(8): 989-992.
13. Zeng W, Liu Z, Li Y, et al. Development and characterization of cores-shell poly (lactide-co-glycolide)-chitosan microparticles for sustained release of GDNF. Colloids Surf B Biointerfaces, 2017, 159: 791-799.
14. Zhang W, Gao Y, Zhou Y, et al. Localized and sustained delivery of erythropoietin from PLGA microspheres promotes functional recovery and nerve regeneration in peripheral nerve injury. Biomed Res Int, 2015, 2015: 478103.
15. Ding T, Zhu C, Yin JB, et al. Slow-releasing rapamycin-coated bionic peripheral nerve scaffold promotes the regeneration of rat sciatic nerve after injury. Life Sci, 2015, 122: 92-99.
16. Xu X, Qiu S, Zhang Y, et al. PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation. Artif Cells Nanomed Biotechnol, 2017, 45(2): 330-339.
17. Li G, Zhao X, Zhang L, et al. Regulating Schwann cells growth by chitosan micropatterning for peripheral nerve regeneration in vitro. Macromol Biosci, 2014, 14(8): 1067-1075.
18. Liu H, Wen W, Hu M, et al. Chitosan conduits combined with nerve growth factor microspheres repair facial nerve defects. Neural Regen Res, 2013, 8(33): 3139-3147.
19. Wang M, Feng Q, Niu X, et al. A spheres-in-sphere structure for improving protein-loading poly (lactide-co-glycolide) microspheres. Polymer Degradation and Stability, 2010, 95(1): 6-13.
20. Tao C, Huang J, Lu Y, et al. Development and characterization of GRGDSPC-modified poly(lactide-co-glycolide acid) porous microspheres incorporated with protein-loaded chitosan microspheres for bone tissue engineering. Colloids Surf B Biointerfaces, 2014, 122: 439-446.
21. Chen MM, Cao H, Liu YY, et al. Sequential delivery of chlorhexidine acetate and bFGF from PLGA-glycol chitosan core-shell microspheres. Colloids Surf B Biointerfaces, 2017, 151: 189-195.

Chinese Journal of Reparative and Reconstructive Surgery

The fabrication and related properties study of chitosan-poly (lactide-co-glycolide) double-walled microspheres loaded with nerve growth factor

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