Cell-penetrating peptides meditated encapsulation of protein therapeutics into intact red blood cells and its application

Abstract

Red blood cells (RBCs) based drug carrier appears to be the most appealing for protein drugs due to their unmatched biocompatability, biodegradability, and long lifespan in the circulation. Numerous methods for encapsulating protein drugs into RBCs were developed, however, most of them induce partial disruption of the cell membrane, resulting in irreversible alterations in both physical and chemical properties of RBCs. Herein, we introduce a novel method for encapsulating proteins into intact RBCs, which was meditated by a cell penetrating peptide (CPP) developed in our lab-low molecular weight protamine (LMWP). l-asparaginase, one of the primary drugs used in treatment of acute lymphoblastic leukemia (ALL), was chosen as a model protein to illustrate the encapsulation into erythrocytes mediated by CPPs. In addition current treatment of ALL using different l-asparaginase delivery and encapsulation methods as well as their associated problems were also reviewed.

Keywords: Acute lymphoblastic leukemia; Cell penetrating peptide; Encapsulation; Erythrocytes; Red blood cells.

Fig. 1

A: The reaction mechanism of L-asparaginase. L-asparaginase is hydrolyzed into L-aspartic acid and ammonia[57]; B: Mechanism of action of L-asparaginase. Figure from Müller and Boos [55].

Fig. 2

Schematic illustration of methods for loading protein drug into erythrocyte. (A) Osmotic dialysis, (B) electroporation, and (C) CPP-mediation method.

Fig. 3

RBC processed by Hypo-osmosis (Top); and Electroporation (Bottom) methods. Left panels: native RBCs with the normal discocyte form; Right panels: processed RBCs with the abnormal stomatocyte form. [80]

Fig. 4

Schematic illustration of the proposed RBC encapsulation technology

Fig. 5

Conversion of asparagine (ASN) to aspartic acid (ASP) by RBC-encapsulated asparaginase (ASNase)

Fig. 6

Confocol microscopic images of untreated RBC (Control RBC; first row); RBC incubated with Alex a Fluor 488-labeled ovalbumin (OVA-488, second row); and LMWP-ovalbumin (LMWP-OVA-488; third row). First column: fluorescence mode; second column: DIC mode; and third column: superimposition. [101]

Fig. 7

Morphological and structural integrity of RBCs after Encapsulation with LMWP-ASNase. Scanning electron microscopic images of (A) Untreated normal RBCs (i.e. Control); and LMWP-ASNase-.encapsulated RBCs; Hematological Parameters of loaded RBCs (B); Osmotic fragility curves for normal (red line) and ASNase-loaded (green line) RBCs (C); oxygen-carrying

Fig. 8

Pharmacokinetic profiles of ASNase activity in blood in DBA2 mice (A) and Kaplan-Meier survival curve for DBA/2 mice bearing L5178Y lymphoma cells (B). [101]