A Novel 3D Printing Particulate Manufacturing Technology for Encapsulation of Protein Therapeutics: Sprayed Multi Adsorbed-Droplet Reposing Technology (SMART)

Abstract

Recently, various innovative technologies have been developed for the enhanced delivery of biologics as attractive formulation targets including polymeric micro and nanoparticles. Combined with personalized medicine, this area can offer a great opportunity for the improvement of therapeutics efficiency and the treatment outcome. Herein, a novel manufacturing method has been introduced to produce protein-loaded chitosan particles with controlled size. This method is based on an additive manufacturing technology that allows for the designing and production of personalized particulate based therapeutic formulations with a precise control over the shape, size, and potentially the geometry. Sprayed multi adsorbed-droplet reposing technology (SMART) consists of the high-pressure extrusion of an ink with a well determined composition using a pneumatic 3D bioprinting approach and flash freezing the extrudate at the printing bed, optionally followed by freeze drying. In the present study, we attempted to manufacture trypsin-loaded chitosan particles using SMART. The ink and products were thoroughly characterized by dynamic light scattering, rheometer, Scanning Electron Microscopy (SEM), and Fourier Transform Infra-Red (FTIR) and Circular Dichroism (CD) spectroscopy. These characterizations confirmed the shape morphology as well as the protein integrity over the process. Further, the effect of various factors on the production were investigated. Our results showed that the concentration of the carrier, chitosan, and the lyoprotectant concentration as well as the extrusion pressure have a significant effect on the particle size. According to CD spectra, SMART ensured Trypsin's secondary structure remained intact regardless of the ink composition and pressure. However, our study revealed that the presence of 5% (w/v) lyoprotectant is essential to maintain the trypsin's proteolytic activity. This study demonstrates, for the first time, the viability of SMART as a single-step efficient process to produce biologics-based stable formulations with a precise control over the particulate morphology which can further be expanded across numerous therapeutic modalities including vaccines and cell/gene therapies.

Keywords: SMART; chitosan nanoparticle; ionic gelation; particle manufacturing; protein encapsulation.

Figure 1

Schematic of Sprayed Multi-Adsorbed Droplet Reposing Technology (SMART).

Figure 2

Effect of chitosan concentration on the ink’s average size. Data points represent mean ± SD (n = 3).

Figure 3

Effect of (A) chitosan concentration, (B) type of lyoprotectant, and (C) concentration of lyoprotectant on flow curves of the inks.

Figure 4

Effect of (A) extrusion pressure, (B) chitosan concentration, (C) trehalose concentration, and (D) type of lyoprotectant on product average diameter size and PDI. Data points represent mean ± SD (n = 3).

Figure 5

SEM images of powdered product.

Figure 6

Fourier-transform infrared spectrums of (A) trypsin, (B) chitosan, and (C) trypsin-loaded chitosan particles made by SMART.

Figure 7

Circular dichroism spectrums of free trypsin and processed and formulated trypsin by SMART.

Figure 8

Effect of (A) lyoprotectant type, (B) lyoprotectant concentration, (C) chitosan concentration, and (D) extrusion pressure on enzyme activity. Data points represent mean ± SD (n = 3). * Significantly different (p < 0.05).

Figure 9

Release kinetics of trypsin from chitosan particles produced by SMART platform. Data points represent mean ± SD (n = 3).