Targeted Delivery of Antisense Oligodeoxynucleotide by Transferrin Conjugated pH-Sensitive Lipopolyplex Nanoparticles: A Novel Oligonucleotide-Based Therapeutic Strategy in Acute Myeloid Leukemia

Abstract

Therapeutic use of oligodeoxynucleotides (ODNs) that hybridize to and downregulate target messenger RNAs (mRNAs) encoding proteins that contribute to malignant transformation has a sound rationale, but has had an overall limited clinical success in cancer due to insufficient intracellular delivery. Here we report a development of formulations capable of promoting targeted delivery and enhanced pharmacologic activity of ODNs in acute myeloid leukemia (AML) cell lines and patient primary cells. In this study, transferrin (Tf) conjugated pH-sensitive lipopolyplex nanoparticles (LPs) were prepared to deliver GTI-2040, an antisense ODN against the R2 subunit of ribonucleotide reductase that has been shown to contribute to chemoresistance in AML. LPs had an average particle size around 110 nm and a moderately positive zeta potential at ~ 10 mV. The ODN encapsulation efficiency of LPs was > 90%. These nanoparticles could release ODNs at acidic endosomal pH and facilitate the cytoplasmic delivery of ODNs after endocytosis. In addition, Tf-mediated targeted delivery of GTI-2040 was achieved. R2 downregulation at both mRNA and protein levels was improved by 8-fold in Kasumi-1 cells and 2 to 20-fold in AML patient primary cells treated with GTI-2040-Tf-LPs, compared to free GTI-2040 treatment. Moreover, Tf-LPs were more effective than non-targeted LPs, with 10 to 100% improvement at various concentrations in Kasumi-1 cells and an average of 45% improvement at 3 µM concentration in AML patient primary cells. Treatment with 1 µM GTI-2040-Tf-LPs sensitized AML cells to the chemotherapy agent cytarabine, by decreasing its IC50 value from 47.69 nM to 9.05 nM. This study suggests that the combination of pH sensitive LP formulation and Tf mediated targeting is a promising strategy for antisense ODN delivery in leukemia therapy.

CD33-Targeted Lipid Nanoparticles (aCD33LNs) for Therapeutic Delivery of GTI-2040 to Acute Myelogenous Leukemia

“…After 4 h incubation at 37 °C, serial diluted Ara-C (0.0001–100 μ M) solutions were added and the cells were incubated for an additional 48 h. MTS solution was then added to each well as described previously. 7 Cell viability was determined by measuring the absorbance of each well at 490 nm on an automated plate reader (Molecular Devices, Sunnyvale, CA, USA). IC 50 values were then calculated based on the measured absorbances.…”

“…5,6 Lipid nanoparticles (LNs) have shown promise as delivery vehicles for ASOs. 7–9 However, further improvement in their effciency is important for their successful application in the clinic. This can be accomplished by further optimization of their composition and by introducing a targeting moiety to facilitate selective delivery to the targeted cell population.…”

A microfluidic method to synthesize transferrin-lipid nanoparticles loaded with siRNA LOR-1284 for therapy of acute myeloid leukemia

“…Tf was conjugated to the surface of NPs by post-insertion and the molar ratio of Tf/lipids was 1/1000 19, 23, 24 . First, human holo-Tf was thiolated at its N-terminus with 2-iminothiolane (Traut’s reagent) in PBS (pH=8.0) and purified by gel filtration on a PD-10 column.…”

“…TfR binding induces internalization of Tf-conjugated nanoparticles via receptor-mediated endocytosis. TfR expression can be upregulated by iron sequestration by a chelator, such as deferoxamine 19 . Tf and antibody to TfR have been evaluated as targeting agents in many published studies for drug, gene, ODN, and siRNA delivery 6, 16, 20 .…”

“…TfR expression can be upregulated by iron sequestration by a chelator, such as deferoxamine 19 . Tf and antibody to TfR have been evaluated as targeting agents in many published studies for drug, gene, ODN, and siRNA delivery 6, 16, 20 . Furthermore, Tf receptor targeted nanoparticles carrying plasmid DNA or ODN have been shown to have enhanced transfection efficiency in Tf-overexpressing tumor cells 21 .…”