High drug-loaded microspheres enabled by controlled in-droplet precipitation promote functional recovery after spinal cord injury

Drug delivery systems with high content of drug can minimize excipients administration, reduce side effects, improve therapeutic efficacy and/or promote patient compliance. However, engineering such systems is extremely challenging, as their loading capacity is inherently limited by the compatibility between drug molecules and carrier materials. To mitigate the drug-carrier compatibility limitation towards therapeutics encapsulation, we developed a sequential solidification strategy. In this strategy, the precisely controlled diffusion of solvents from droplets ensures the fast in-droplet precipitation of drug molecules prior to the solidification of polymer materials. After polymer solidification, a mass of drug nanoparticles is embedded in the polymer matrix, forming a nano-in-micro structured microsphere. All the obtained microspheres exhibit long-term storage stability, controlled release of drug molecules, and most importantly, high mass fraction of therapeutics (21.8–63.1 wt%). Benefiting from their high drug loading degree, the nano-in-micro structured acetalated dextran microspheres deliver a high dose of methylprednisolone (400 μg) within the limited administration volume (10 μL) by one single intrathecal injection. The amount of acetalated dextran used was 1/433 of that of low drug-loaded microspheres. Moreover, the controlled release of methylprednisolone from high drug-loaded microspheres contributes to improved therapeutic efficacy and reduced side effects than low drug-loaded microspheres and free drug in spinal cord injury therapy.

HMPS@AcDX group was compared with LAcDX group (  ); statistical significance was analyzed using one-way ANOVA followed by Fisher's post-hoc test. Delayed treatment group was compared with immediate treatment group ( # ); statistical significance was analyzed using pair-sample t-test. ** ,  P < 0.01, *** ,  P < 0.001. Exact P values are given in the Source Data file. For both immediate treatment and delayed treatment, throughout the entire assessment period, HMPS@AcDX recovered motor function significantly faster than the other groups including LAcDX. In addition, for HMPS@AcDX, there was no significant difference in the BBB score between immediate treatment and delayed treatment. Data are presented as mean values ± SD. S17 HMPS@AcDX group was compared with LAcDX group (  ); statistical significance was analyzed using one-way ANOVA followed by Fisher's post-hoc test. Delayed treatment group was compared with immediate treatment group ( # ); statistical significance was analyzed using pair-sample t-test.  P < 0.01, *** ,  P < 0.001. Exact P values are given in the Source Data file. Four weeks after injury, the lesion volume in the groups treated by LAcDX (P < 0.001) and HMPS@AcDX (P < 0.001) was notably smaller than that of the SCI group for both immediate treatment and delayed treatment. In comparison with LAcDX, HMPS@AcDX significantly reduced the loss of post-traumatic spinal cord tissue (P < 0.001 for immediate treatment and P < 0.01 for delayed treatment). In addition, for HMPS@AcDX, there was no significant difference in the lesion volume between immediate treatment and delayed treatment. S18 Box plots show the minimum value, the first quartile, the median, the third quartile, and the maximum value. The intervention groups were compared with the SCI group ( * ); HMPS@AcDX group was compared with LAcDX group (  ); statistical significance was analyzed using one-way ANOVA followed by Fisher's post-hoc test. Delayed treatment group was compared with immediate treatment group ( # ); statistical significance was analyzed using pair-sample t-test.  P < 0.05, ** ,  P < 0.01, *** ,  P < 0.001. Exact P values are given in the Source Data file. As compared to SCI group, the treatment with LAcDX (P < 0.01) and HMPS@AcDX (P < 0.001) significantly inhibited the increase of GFAP immunoreactivity for both immediate treatment and delayed treatment. In comparison with LAcDX, HMPS@AcDX significantly inhibited the increase of GFAP immunoreactivity (P < 0.01 for immediate treatment and P < 0.05 for delayed treatment). In addition, for HMPS@AcDX, there was no significant difference in the GFAP immunoreactivity between immediate treatment and delayed treatment. The density of CD68-positive microglia in the lesion area for LAcDX was significantly smaller than that in SCI group (P < 0.001 for both immediate treatment and delayed treatment). HMPS@AcDX further significantly reduced the number of microglia in the traumatic lesion area compared to LAcDX group (P < 0.001 for both immediate treatment and S19 delayed treatment). In addition, for HMPS@AcDX, there was no significant difference in the density of CD68-positive microglia between immediate treatment and delayed treatment.
S20 intervention groups were compared with the SCI group ( * ); HMPS@AcDX group was compared with LAcDX group (  ); statistical significance was analyzed using one-way ANOVA followed by Fisher's post-hoc test. Delayed treatment group was compared with immediate treatment group ( # ); statistical significance was analyzed using pair-sample t-test.  P < 0.01, *** ,  P < 0.001. Exact P values are given in the Source Data file. The CSPG level for LAcDX was significantly smaller than that in SCI group (P < 0.001 for both immediate treatment and delayed treatment). HMPS@AcDX further significantly reduced the CSPG level compared to LAcDX group (P < 0.01 for immediate treatment and P < 0.001 for delayed treatment). In addition, for HMPS@AcDX, there was no significant difference in the CSPG level between immediate treatment and delayed treatment. intervention groups were compared with the SCI group ( * ); HMPS@AcDX group was compared with LAcDX group (  ); statistical significance was analyzed using one-way ANOVA followed by Fisher's post-hoc test. Delayed treatment group was compared with immediate treatment group ( # ); statistical significance was analyzed using pair-sample t-test.  P < 0.01, *** ,  P < 0.001. Exact P values are given in the Source Data file. For both immediate treatment and delayed treatment, in comparison with SCI group, a significant decrease in NF200 intensity was observed for LAcDX (P < 0.001) and HMPS@AcDX (P < 0.001) groups.
HMPS@AcDX further significantly reduced the NF200 intensity compared to LAcDX group (P < 0.01 for both immediate treatment and delayed treatment). In addition, for HMPS@AcDX, there was no significant difference in the NF200 intensity between immediate treatment and delayed treatment. In comparison with SCI group, a significant decrease in MBP intensity was observed for LAcDX (P < 0.001 for both immediate treatment and delayed treatment) and HMPS@AcDX (P < 0.001 for both immediate treatment and delayed treatment) groups.
HMPS@AcDX further significantly reduced the MBP intensity compared to LAcDX group (P < 0.001 for immediate treatment and P < 0.01 for delayed treatment). In addition, for S22 HMPS@AcDX, there was no significant difference in the MBP intensity between immediate treatment and delayed treatment.
S23 Fig. S20. Nuclear magnetic resonance spectra of the dextran and the synthesized AcDX.  Table S1. Simulated and experimental loading degree, encapsulation efficiency and particle size of HAcDX@MPS microspheres. Data are presented as mean values ± SD.

Method
Loading degree (wt %) Encapsulation efficiency (%) Particle size ( Note: Microspheres were prepared using inner fluid consisting of 10 mg/mL AcDX and 30 mg/mL MPS in a mixture of dimethyl sulfoxide and ethyl acetate (1:9, v/v). Table S2. The mass of samples and volume of release media for drug release studies.

S25
Sample name Mass of sample (mg) Volume of release media (mL)