pH-Responsive Polysaccharide-Based Polyelectrolyte Complexes As Nanocarriers for Lysosomal Delivery of Therapeutic Proteins

Abstract

Nanopharmaceutics composed of a carrier and a protein have the potential to improve the activity of therapeutical proteins. Therapy for lysosomal diseases is limited by the lack of effective protein delivery systems that allow the controlled release of specific proteins to the lysosomes. Here we address this problem by developing functional polyelectrolyte-based nanoparticles able to promote acidic pH-triggered release of the loaded protein. Trimethyl chitosan (TMC) was synthesized and allowed to form polyelectrolyte complexes (PECs) with the lysosomal enzyme α-GAL through self-assembly and ionotropic gelation, with average particle size <200 nm, polydispersity index (PDI) <0.2, ζ potential of ∼ 20 mV, and a protein loading efficiency close to 65%. These polyelectrolyte nanoparticles were stable and active under physiological conditions and able to release the enzyme at acidic pH, as demonstrated by in situ atomic force microscopy (AFM). These nanoparticles were further functionalized with Atto 647N for single-particle characterization and tracking their cellular uptake and fate using high-resolution fluorescence microscopy. In contrast with their precursor, TMC, PECs were efficiently internalized by human endothelial cells and mostly accumulated in lysosomal compartments. The superior physicochemical characteristics of the TMC/α-GAL PECs together with their excellent cellular uptake properties indicate their enormous potential as advanced protein delivery systems for the treatment of lysosomal storage diseases.

Highly Versatile Polyelectrolyte Complexes for Improving the Enzyme Replacement Therapy of Lysosomal Storage Disorders

“…5 via a two-step procedure, 28 as reported 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 7 previously. 5 Briefly, a mixture of 2 g of sieved chitosan, 4.8 g of sodium iodide (NaI), 11 mL of a 15% aqueous sodium hydroxide (NaOH) solution and 11.5 mL of methyl iodide (CH 3 I) in 80 mL of 1-methyl-2-pyrrolidinone was stirred at 60 °C for 1 h.…”

“…33 Polyelectrolyte complexes (PECs) between GLA and TMC were prepared by ionotropic gelation with penta-sodium triphosphate (TPP, Fluka). 5 All PECs were characterized and used as obtained, without further purification or separation steps. When necessary, they were stored at 4 ºC.…”

“…4 Within the context of protein delivery systems, stimulitriggered release is an appealing and promising approach, especially with the use of physiological stimuli like the pH drop within endo-lysosomal compartments. 5,6 In addition, addressing clinically relevant end points by targeted delivery has emerged as an approach to maximize the efficiency of a therapeutic macromolecule by strictly 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 5 localizing its pharmacological activity through controlled biodistribution. 7,8 Drug delivery to the nucleus or cytoplasm has been extensively explored, 9 however, lysosomal delivery has only lately turned into the focus of attention.…”

“…7,8 Drug delivery to the nucleus or cytoplasm has been extensively explored, 9 however, lysosomal delivery has only lately turned into the focus of attention. 5,[10][11][12][13][14][15][16][17] The current ERT therapy relies in the natural cellular transit of lysosomal enzymes.…”

“…[24][25][26][27] Previously, we have described for the first time the development of a pH-responsive nanocarrier for lysosomal delivery of proteins, based on the ionotropic gelation of complexes between trimethyl chitosan (TMC) and the lysosomal enzyme GLA, with triphosphate ions. 5 With a good protein loading efficiency (around 65%), these polyelectrolyte complexes (PECs) have appropriate physicochemical properties to remain stable at physiological temperature and pH (around 7.5), and dissociate and release the protein content at endosomal/lysosomal values (pH 4.5 to 5.5). We proved that these PECs were efficiently internalized by human cells and mostly accumulated in lysosomal compartments, demonstrating their enormous potential as advanced protein delivery systems for treatment of lysosomal storage diseases.…”