To determine the stability and surface charge of chitosan-hydroxyapatite nanoparticles, the particles were incubated at 4°C for 4 weeks. The sizes of chitosan-hydroxyapatite nanoparticles were around 105 nm and the zeta potential of chitosan-hydroxyapatite nanoparticles was found to be nearly 9 mV, which suggested that the stability of the particles was successfully maintained at low temperature for 4 weeks (Table 1). In addition, the sizes of chitosan nanoparticles were around 90 nm and their zeta potential were nearly 11 mV at 4°C in 4 weeks (Table 1).

The modification of chitosan-hydroxyapatite nanoparticles was confirmed by FTIR analysis. As shown in Figure 1A, hydroxyapatite was successful attached to chitosan nanoparticles. For chitosan-hydroxyapatite nanoparticles, the spectrum showed characteristic absorption of peak around 3,500 cm⁻¹ which was attributed to the combined stretching vibration of OH and N-H groups, whereas at 1,700 cm⁻¹ was the characteristic peaks of amide. Moreover, C-H stretching of rock was observed at 1,400 cm⁻¹, and C-O-C stretching mode at 1,150 and 1,050 cm⁻¹. The spectra of peak around 1,070 and 570 cm⁻¹ represented PO43− stretching group, and the peak of 1,450 cm⁻¹ indicated the CO32− stretching vibration, which were the characteristic absorption bands of hydroxyapatite, demonstrating that hydroxyapatite was successfully coated in chitosan nanoparticles. Low-molecular weight chitosan-hydroxyapatite nanoparticles were studied under SEM for the integrity (Figure 1B). It was observed that hydroxyapatite appeared to have a mixture of fine micro and nano granules that were integrated with the chitosan scaffold (Figure 1B).

To investigate the effect of chitosan-hydroxyapatite nanoparticles on targeted delivery to the injured spinal cord, the Cy5.5 was labeled to chitosan-hydroxyapatite nanoparticles at room temperature. The Cy5.5 labeled chitosan-hydroxyapatite and chitosan were used to treat the rats of SCI by intravenous administration and quantified in the injured spinal cord at 48 h after SCI (Figure 2A). The concentration of the particles was measured by detecting the fluorescence intensity of Cy5.5. The result demonstrated that the efficiency of delivery to the injured spinal cord were significantly enhanced by the treatment of chitosan-hydroxyapatite compared with chitosan treatment group, which was estimated through the fluorescence intensity of Cy5.5 at the injured spinal cord (Figure 2B).

To investigate the effect of chitosan-hydroxyapatite on SCI, we assessed the function repair by treatment of chitosan-hydroxyapatite after SCI. The BBB scores were significantly increased at 7 days after treatment of chitosan-hydroxyapatite, compared with the control SCI group (Figure 3A). On the other hand, chitosan alone treatment resulted in no significant increase of the BBB scores at different time points compared with the control group (Figure 3A). In order to explore the effect of chitosan-hydroxyapatite on tissue recovery after SCI, the HE staining was performed at 28 days after injury. The result demonstrated that administration of chitosan-hydroxyapatite significantly reduced the lesion cavity volume, and chitosan treatment resulted in no significant improvement of the lesion cavity volume compared the SCI group (Figure 3B). In addition, the dispersed structure was apparently improved by the chitosan-hydroxyapatite treatment when compared with the SCI and CN treatment group (Figure 3B).

Hydroxyapatite was prepared and characterized by Fourier Transform infrared spectroscopy (FT-IR) according to the reported methods (9). A total of 2.5 g of low-molecular weight chitosan (1,526 Da) was dissolved in 250 mL of 2% acetic acid solution. The solution was stirred overnight on a mechanical stirrer (RW 20.n Labortechik) and sonicated for 1 h to remove any air bubbles. The solution was lyophilized in freeze dryer to form chitosan scaffolds. A total of 2.5 g of prepared hydroxyapatite was suspended in 50 mL of water and carefully transferred into the chitosan solution. The solution was mechanically stirred for 24 h to disperse the hydroxyapatite particles in the polymer matrix in a homogeneous manner. The mixed solution was lyophilized in freeze dryer to form chitosan-hydroxyapatite nanoparticles.

Chitosan-hydroxyapatite nanoparticles samples were prepared by drop drying a water diluted suspension. The mean diameter of the studied samples was determined by statistical analysis. The surface charges of nanoparticles in distilled water were determined using a zeta potential analyzer (the measurement temperature at 25°C), (Brookhaven Instrument Co., CA). The nanoparticles size was characterized through dynamic light scattering (He-Ne laser, wavelength 633 nm, incident Angle 90°, set the measurement temperature at 25°C, time for 5 min, the nanoparticles were placed in the particle size pool Appropriate amount of nanoparticles in the particle size pool) (DLS).

FTIR was used to evaluate the modification of chitosan-hydroxyapatite nanoparticles. The chitosan-hydroxyapatite nanoparticles were recovered by freeze-drying and characterized by FTIR spectroscopic analysis. Briefly, 500 μg nanoparticle powder was mixed and ground with spectroscopic-grade KBr. IR spectra was recorded using an FTIR spectrophotometer (Thermo, USA) within the range of 500–4,000 cm⁻¹. Chitosan nanoparticles were determined as control.

The structural integrity of chitosan-hydroxyapatite was analyzed and the image was detected by Scanning Electron Microscopy (SEM, HITACHI, Japan). Chitosan-hydroxyapatite and chitosan were fixed overnight in the buffer containing 25 g/L glutaraldehyde (glutaraldehyde was dissolved in 0.1 mol/L sodium diarsonic acid buffer), respectively, and dehydrated with gradient ethanol. The treated samples were freezed for 2 d and Metal spraying.

Cy5.5 was dissolved in DMSO, added to chitosan or chitosan-hydroxyapatite solution and left at room temperature in the dark for 6 h. The solution was performed with dialysis against distilled water. The amounts of Cy5.5 in the chitosan or chitosan-hydroxyapatite treatment in the injured spinal cord were determined by fluorescence.

Adult wistar-rats (males) are 6 weeks old [(180–220 g), Sprague-Dawley, Harlan] were provided by the Animal Center of Second Military Medical University. All of the animals were treated humanely and with regard for the alleviation of suffering. This study was carried out in accordance with the guidelines of the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental protocols described in this study were approved by the Ethics Review Committee for Animal Experimentation of the Second Military Medical University. The permission number for animal experiment is SHOU-DW-2021-074.

Allen's method was used to make SCI rat model. The rats were anesthetized with 4% isoflurane. A laminectomy was performed at the thoracic vertebra level 10 (T10) after shaving and cleaning until fully recovered from the anesthesia. Spinal cord contusion was induced using a weight-drop apparatus, where a guided 5 g rod was dropped from a height of 80 mm onto the exposed cord, representing moderate SCI. After surgery, the muscles were sutured in layers and the skin incision was closed with silk threads. Penicillin G (40,000 U, i.m.) was administrated daily for 3 days to prevent infection.

Twelve rats were randomly assigned to three groups: SCI rats (n = 4), chitosan-treated SCI rats (n = 4), chitosan-hydroxyapatite-treated SCI rats (n = 4). 15 mg/kg concentrations of chitosan-hydroxyapatite and chitosan were intravenously administered daily for 5 days and started at 1 h after injury. After injury, the mice in the SCI group were injected with saline solution through the tail vein. The other groups were administrated with 15 mg/kg concentration of chitosan-hydroxyapatite, 15 mg/kg concentration of chitosan (500 μL in saline) through a single intravenous tail vein injection.

Cy5.5 was dissolved in DMSO and added to chitosan or chitosan-hydroxyapatite solution for 6 h at room temperature in the dark. The solution was performed with dialysis against distilled water. The content of Cy5.5 in the labeled chitosan or chitosan-hydroxyapatite was determined using fluorescence method to ensure that the two samples had the same labeled density and were comparable. Briefly, 15 mg/kg concentrations of Cy5.5 labeled chitosan-hydroxyapatite and chitosan nanoparticles were intravenously administered in SCI rats at 1 h post-injury, respectively. At 48 h after the injection, the rats were sacrificed and the tissues of injured spinal cord were harvested. The cell nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI), respectively. The collected spinal cord tissue was placed on a slide, stained with 15 μLDAPI, and observed after 15 min. The fluorescence images of Cy5.5 labeled to chitosan-hydroxyapatite and chitosan nanoparticles in the injured spinal cord were captured with a laser confocal microscope (Leica).

The locomotor activity was assessed at 1, 3, 7, 14, 21, 28 days post-injury using the Basso Beattie Bresnahan (BBB) locomotor score method. The rats were put into an open box, and the wall of the box was tapping to make them crawl. The movement distance, hip, knee and ankle walking, trunk movement and coordination of the animals were observed. Each rat was observed for 15 min, and the movement ability of the rats in each group was tested by BBB score. The final score for each animal was obtained by averaging values from both investigators.

The cavity volume and radiated structure were evaluated by HE staining. After the last injection, the rats were anesthetized to death, and 2 cm of damaged spinal cord was extracted, and the spinal cord was treated with phosphate buffer (PBS) washing. The 5 μm longitudinal sections were made from the paraffin embedded blocks and stained with hematoxylin solution for 5 min. Then the sections were stained with eosin solution for 3 min and followed by dehydration with graded alcohol and clearing in xylene. The mounted slides were then observed and photographed using a light microscope (Nikon, Tokyo, Japan).

The data are expressed as the means ± standard deviations of three independent experiments. The statistical differences were calculated by the Student's t-test or one-way ANOVA analysis of variance with Dunnett's test. The data were considered statistically significant when values reached P < 0.05.