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

Abstract

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.

Fig. 1. Preparation of nano-in-micro structured microspheres.

a The preparation of high drug-loaded microspheres by droplet microfluidics. b The structural evolution of the droplets during solidification. c Propeller-shaped TPE is non-emissive when dissolved but becomes highly emissive when its molecules are aggregated, due to the restriction of intramolecular rotation of its phenyl rotors against its ethylene stator in the aggregate state. d Droplets generated by flow-focusing microfluidic device pass through the spiral-shaped capillary tube under ultraviolet irradiation. e Scanning electron microscope images of whole and enlarged cross-section of TPE@AcDX microspheres. X-ray powder diffraction (f) and differential scanning calorimetry (g) of TPE@AcDX microspheres and TPE.

Fig. 2. The versatility of controlled in-droplet precipitation.

a Light microscope images of drug precipitation process in the droplets generated by co-flow microfluidics. b, c Scanning electron microscope images of microspheres prepared by droplet microfluidics, using AcDX (b) and PLGA (c) as carrier matrix. d, e Drug loading degree (d) and encapsulation efficiency (e) of microspheres prepared with and without dimethyl sulfoxide in the inner fluid (n = 3). Data are presented as mean values ± SD. f, g X-ray powder diffractogram (f) and differential scanning calorimetry curves (g) show the solid-state of the encapsulated drug molecules in microspheres.

Fig. 3. Numerical simulation of the droplet solidification and drug encapsulation.

a Concentration fields of MPS on the mid-plane of the droplet. The color bar on the right side represents the concentration magnitude of MPS. b The shrinking of a droplet as a function of time during the solidification process. c Nominal concentration (mass of drug divided by volume of droplet) of MPS at different states (dissolved and precipitated) in the droplet during the solidification process. d Transmission electron microscope images of thin section of bare AcDX, MPS@AcDX, and washed thin section of MPS@AcDX. e The impact of initial dimethyl sulfoxide ratio in the inner fluid on the simulated diffusion time that taken for MPS to reach the saturation concentration in droplets. f, g Numerically simulated (f) and experimental (g; n = 3) loading degree and encapsulation efficiency of AcDX microspheres prepared by varying the dimethyl sulfoxide ratio in the inner fluid. AcDX concentration and MPS concentration are set at 10 mg/mL and 30 mg/mL, respectively. Data are presented as mean values ± SD. h The impact of initial MPS concentration in the inner fluid on the numerically simulated diffusion time that taken for MPS to reach saturation concentration in the droplets. i, j Numerically simulated (i) and experimental (j; n = 3) loading degree and encapsulation efficiency of AcDX microspheres. AcDX concentration and dimethyl sulfoxide ratio are set at 10 mg/mL and 10% v/v, respectively. Data are presented as mean values ± SD.

Fig. 4. Release profiles and stability of the prepared microspheres.

a, b Release profiles of ATV, MPS, or HCT from AcDX (a) or PLGA (b) microspheres (n = 3). Data are presented as mean values ± SD. c–e X-ray powder diffractogram of drug-loaded AcDX microspheres, ATV@AcDX (c), MPS@AcDX (d) and HCT@AcDX (e), stored at 25 °C/40% relative humidity for 6 and 12 months. f–h X-ray powder diffractogram of drug-loaded PLGA microspheres, ATV@PLGA (f), MPS@PLGA (g), and HCT@PLGA (h), stored at 25 °C/40% relative humidity for 6 and 12 months.

Fig. 5. HMPS@AcDX microspheres improve motor function and reduce spinal cord edema.

a Microspheres were intrathecally injected within 5 min after weight drop at T10 level. b–d MPS concentration in cerebral spinal fluid (CSF) as a function of time (b), and the corresponding area under curve (AUC; c) and mean residence time (MRT; d). n = 5 rats per time point; every rat was only sampled once. e Rats were functionally graded up to 28 days post-injury by BBB grading scale (n = 10, 7, 8, and 9 for SCI/MPS/LAcDX, HAcDX, LMPS@AcDX and HMPS@AcDX, respectively. Data are presented as mean values ± SD for (b–e). f Sagittal spinal cord sections stained with Nissl display the injury area of spinal cord at day-28 post trauma. g Comparison of the lesion volume after treatment (n = 5 per group). h Sagittal and axial spinal cord T2 weighted images at day-28 after injury. (1) & (1′) are axial images in injury epicenter, (2) & (2′) are axial images in far-injury area, (1′) & (2′) are axial color maps transformed from (1) and (2) normal axial images and the blue color represents the edema signal. i Intensity ratio for the signal of lesion to normal spinal cord and cord volume determined for consecutive slices at day-1 and day-28 post injury (n = 5 per group). j TUNEL-positive apoptotic cells (in green) in sagittal spinal cord sections at day-1 post trauma. The nuclei of all cells were stained with DAPI (in blue). k Comparison of the number of TUNEL-positive cells after treatment (n = 6 per group). Box plots show the minimum value, the first quartile, the median, the third quartile, and the maximum value. The intervention groups were compared with SCI group (*); HMPS@AcDX group was compared with LMPS@AcDX group (^#) and LAcDX group (^⊥); *, ^⊥P < 0.05, **, ^##P < 0.01, ***, ^###, ^⊥⊥⊥P < 0.001. Statistical significance was analyzed using one-way ANOVA followed by Fisher’s post-hoc test. Exact P values are given in the Source data file.

Fig. 6. HMPS@AcDX inhibits gliosis, attenuates the activation of microglia, suppresses CSGP production, and protects axon and myelin sheath.

a Representative immunohistochemical staining of GFAP (in green) and CD68 (in red) in longitudinal sections of injured spinal cord at day-28 post injury. The nuclei of all cells were stained with DAPI (in blue). b Semi-quantification of GFAP intensity and density of microglia in the injured spinal cord. For GFAP intensity, the data are plotted as the relative ratio of the immunoreactivity near the injury site compared with that in distant area. (n = 6 per group). c Representative immunohistochemical staining of GFAP (in green) and CD68 (in red) adjacent to the lesion area. d The deposition of chondroitin sulfate proteoglycans (CSPGs) determined by CS56 antibody (in red) at day-28 after injury. e Semi-quantification of CS56 intensity increase in the traumatic lesion area. The data are plotted as the relative ratio of the immunoreactivity near the injury site compared with that in the distant area. (n = 6 per group). f Enlarged immunohistochemical staining of GFAP (in green) and CS56 (in red) adjacent to the lesion area. g Immunohistochemical staining of MBP (in green) and NF200 (in red) in injured spinal cord at day-28 post trauma. h Semi-quantification of NF200 and MBP intensity after spinal cord injury therapy. The data are plotted as the relative ratio of the immunoreactivity near the injury site compared with that in distant area. (n = 6 per group). Box plots show the minimum value, the first quartile, the median, the third quartile, and the maximum value. i Representative immunohistochemical staining of MBP (in green) and NF200 (in red) adjacent to the lesion area. The intervention groups were compared with the SCI group (*); HMPS@AcDX group was compared with the LMPS@AcDX group (^#) and LAcDX group (^⊥); *, ^⊥ P < 0.05, **, ^##, ^⊥⊥ P < 0.01, ***, ^### P < 0.001. Statistical significance was analyzed using one-way ANOVA followed by Fisher’s post-hoc test. Exact P values are given in the Source data file.