Hyaluronic Acid-Based Nanoparticles for Protein Delivery: Systematic Examination of Microfluidic Production Conditions

Abstract

Hyaluronic acid-based nanoparticles (HA NPs) can be used to deliver a protein cargo to cells overexpressing HA receptors such as CD44 since they combine the low toxicity of the carrier and the retention of the protein integrity with the receptor-mediated internalization. HA properties play a crucial but sometimes unclear role in managing the formation and stability of the meshwork, cell interactions, and ultimately the protein entrapment efficacy. Nowadays, microfluidic is an innovative technology that allows to overcome limits linked to the NPs production, guaranteeing reproducibility and control of individual batches. Taking advantage of this technique, in this research work, the role of HA weight average molecular weight (Mw) in NPs formation inside a microfluidic device has been specifically faced. Based on the relationship between polymer Mw and solution viscosity, a methodological approach has been proposed to ensure critical quality attributes (size of 200 nm, PDI ≤ 0.3) to NPs made by HA with different Mw (280, 540, 710 and 820 kDa). The feasibility of the protein encapsulation was demonstrated by using Myoglobin, as a model neutral protein, with an encapsulation efficiency always higher than 50%. Lastly, all NPs samples were successfully internalized by CD44-expressing cells.

CD44-Targeted Carriers: The Role of Molecular Weight of Hyaluronic Acid in the Uptake of Hyaluronic Acid-Based Nanoparticles

“…HA/TPP and CS aqueous solutions were pumped into separate inlets of the microfluidic device. As demonstrated by our previous work [27], depending on the HA Mw, the HA and CS concentration were properly changed as reported in Table 1. The TPP concentration was kept constant at 2 µg/mL, and it was used as ancillary crosslinker to trigger the CS nucleation.…”

“…Aiming to evaluate only the effect of HA Mw on the NPs' uptake, NPs with comparable physical features were produced by HA having different Mw, namely, 280 kDa, 540 kDa and 820 kDa. By exploiting the methodological approach proposed in our previous work [27], the recovered HA/CS NPs showed particles size of around 200 nm, a PDI < 0.3 and negative surface charges. The size and size uniformity agree with the most common nanomedicine applications, meanwhile, the negative surface charge indicates the presence of HA on the NPs surface that is crucial for the CD44 targeting.…”

“…HA/CS NPs were prepared by one-step polyelectrolyte complexation through a microfluidic device equipped with an SHM micromixer. Please refer to our previous paper for a detailed study of microfluidic production conditions [27]. The HA/CS NPs made of HA with an Mw of 280 kDa presented a mean size of 211 ± 19.3 nm with a PDI of 0.30 ± 0.02 and a negatively charged surface (−16.6 ± 2.21 mV).…”

“…In our previous work, HA/CS NPs were successfully produced by microfluidic technology, a relatively new technology for the reproducible and tunable synthesis of NPs for biomedical purposes [26,27]. A microfluidics system is a straightforward and effective tool for the synthesis of nanomaterials, ensuring a precise control over size, shape, and chemical composition through the manipulation of fluids in a microchannel.…”

“…Then, the role of HA Mw in the NPs' assembly into the microfluidic channel has been specifically faced. Finally, a relationship between polymer Mw and solution viscosity has been proposed to maintain specific quality attributes (size, PDI, surface charge) to NPs [26,27]. The feasibility and reliability of the HA/CS NPs as a drug delivery system have been proven by our research group for different payloads, both in lipophilic synthetic drugs (Everolimus) and macromolecules (Myoglobin).…”