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

Abstract

The siRNA LOR-1284 targets the R2 subunit of ribonucleotide reductase (RRM2) and has shown promise in cancer therapy. In this study, transferrin (Tf) conjugated lipid nanoparticles (Tf-NP-LOR-1284) were synthesized by microfluidic hydrodynamic focusing (MHF) and evaluated for the targeted delivery of LOR-1284 siRNA into acute myeloid leukemia (AML) cells. The in vitro study showed that Tf-NP-LOR-1284 can protect LOR-1284 from serum nuclease degradation. Selective uptake of Tf-NP-LOR-1284 was observed in MV4-11 cells. In addition, qRT-PCR and Western blot results revealed that Tf-NP-LOR-1284 was more effective than the free LOR-1284 in reducing the R2 mRNA and protein levels. The Tf-NP-LOR-1284 showed prolonged circulation time and increased AUC after i.v. administration relative to the free LOR-1284. Furthermore, Tf-NP-LOR-1284 facilitated increased accumulation at the tumor site along with the decreased R2 mRNA and protein expression in a murine xenograft model. These results suggest that Tf-conjugated NPs prepared by MHF provide a suitable platform for efficient and specific therapeutic delivery of LOR-1284 into AML cells.

Figure 1. Synthesis of siRNA-loaded NPs by MHF

(A) Photographs of the MF chip. The device consisted of three inlet ports and one outlet port. The inlet ports were connected to sterile syringes containing either cationic NPs or siRNA buffer solution. (B) Schematics for flow patterns T1 and T2. The effects of flow patterns and flow rates on the particle size of Tf-NP-siRNA for DC-Chol based NPs (C) and DOTMA, DODMA based NPs (D), respectively. Results are presented as mean ± SD of three independent experiments.

Figure 2. In vitro evaluation of Tf-NP-siRNA

(A) The stability of Tf-NP-siRNA in serum. (B) Stability of Tf-NPs on siRNA in serum by agarose gel electrophoresis. The lanes are: 1. free siRNA without serum incubation. 2–5. Tf-NP-siRNA incubated for 0.5, 1, 2, or 4 h in serum. 6–9. free siRNA incubated for 0.5, 1, 2, or 4 h in serum. The bands above the siRNA bands in lanes 2–9 were due to SDS micelles. (C) Cytotoxicity study of Tf-NP-siRNA on MV4–11 by MTS. MV4–11 cells were transfected by Tf-NP-NC-siRNA, free siRNA, or Tf-NP-siRNA for 48 h. Results are presented as mean ± SD of three independent experiments.

Figure 2. In vitro evaluation of Tf-NP-siRNA

(A) The stability of Tf-NP-siRNA in serum. (B) Stability of Tf-NPs on siRNA in serum by agarose gel electrophoresis. The lanes are: 1. free siRNA without serum incubation. 2–5. Tf-NP-siRNA incubated for 0.5, 1, 2, or 4 h in serum. 6–9. free siRNA incubated for 0.5, 1, 2, or 4 h in serum. The bands above the siRNA bands in lanes 2–9 were due to SDS micelles. (C) Cytotoxicity study of Tf-NP-siRNA on MV4–11 by MTS. MV4–11 cells were transfected by Tf-NP-NC-siRNA, free siRNA, or Tf-NP-siRNA for 48 h. Results are presented as mean ± SD of three independent experiments.

Figure 2. In vitro evaluation of Tf-NP-siRNA

(A) The stability of Tf-NP-siRNA in serum. (B) Stability of Tf-NPs on siRNA in serum by agarose gel electrophoresis. The lanes are: 1. free siRNA without serum incubation. 2–5. Tf-NP-siRNA incubated for 0.5, 1, 2, or 4 h in serum. 6–9. free siRNA incubated for 0.5, 1, 2, or 4 h in serum. The bands above the siRNA bands in lanes 2–9 were due to SDS micelles. (C) Cytotoxicity study of Tf-NP-siRNA on MV4–11 by MTS. MV4–11 cells were transfected by Tf-NP-NC-siRNA, free siRNA, or Tf-NP-siRNA for 48 h. Results are presented as mean ± SD of three independent experiments.

Figure 3. Cellular uptake and intracellular location of Tf-NP-siRNA in MV4–11 cells

(A) Flow cytometry analysis on expression levels of TfR (also known as CD71) on the surface of AML cells. Cells were surface stained with PE-labeled anti-TfR (anti-CD71) monoclonal antibodies (BD Biosciences, San Jose, CA) for 30min on ice, followed by flow cytometry analysis. (B) Cellular uptake of FAM-labeled Tf-NP-siRNA by flow cytometry. (C) Intracellular localization of Tf-NP-siRNA 4 h after transfection by confocal microscopy.

Figure 3. Cellular uptake and intracellular location of Tf-NP-siRNA in MV4–11 cells

(A) Flow cytometry analysis on expression levels of TfR (also known as CD71) on the surface of AML cells. Cells were surface stained with PE-labeled anti-TfR (anti-CD71) monoclonal antibodies (BD Biosciences, San Jose, CA) for 30min on ice, followed by flow cytometry analysis. (B) Cellular uptake of FAM-labeled Tf-NP-siRNA by flow cytometry. (C) Intracellular localization of Tf-NP-siRNA 4 h after transfection by confocal microscopy.

Figure 3. Cellular uptake and intracellular location of Tf-NP-siRNA in MV4–11 cells

(A) Flow cytometry analysis on expression levels of TfR (also known as CD71) on the surface of AML cells. Cells were surface stained with PE-labeled anti-TfR (anti-CD71) monoclonal antibodies (BD Biosciences, San Jose, CA) for 30min on ice, followed by flow cytometry analysis. (B) Cellular uptake of FAM-labeled Tf-NP-siRNA by flow cytometry. (C) Intracellular localization of Tf-NP-siRNA 4 h after transfection by confocal microscopy.

Figure 4. Tf-NP-siRNA mediated gene silencing in MV4–11 and K562 cells

(A) Concentration-dependent effect of Tf-NP-siRNA on R2 mRNA knockdown by qRT-PCR. (B) The effect of Tf-NP-siRNA on downregulation of R2 protein relative to control groups by Western blot. Cells were transfected by Tf-NP-NC-siRNA (200 nM) or Tf-NP-siRNA (200 nM) for 48 h.

Figure 4. Tf-NP-siRNA mediated gene silencing in MV4–11 and K562 cells

(A) Concentration-dependent effect of Tf-NP-siRNA on R2 mRNA knockdown by qRT-PCR. (B) The effect of Tf-NP-siRNA on downregulation of R2 protein relative to control groups by Western blot. Cells were transfected by Tf-NP-NC-siRNA (200 nM) or Tf-NP-siRNA (200 nM) for 48 h.

Figure 5. Pharmacokinetic study of Cy3 labeled Tf-NP-siRNA in ICR mice

(A) The standard curve of MFI versus the concentration of Cy3-siRNA. (B) The plasma concentrations of Cy3-labeled Tf-NP-siRNA and free Cy3-siRNA after injection. Data represents the mean ± SD (n=3).

Figure 6. In vivo distribution study of Cy5 labeled Tf-NP-siRNA

(A) Tf-mediated tumor targeting distribution of Cy5-siRNA by IVIS imaging. 1–2: Cy5-labeled NP-siRNA in NOD-SCID mice. 3: Cy5-labeled NP-siRNA in normal SCID mice. 4–5: Cy5-labeled Tf-NP-siRNA in NOD-SCID mice. 3: Cy5-labeled Tf-NP-siRNA in normal SCID mice. (B) Cy5-labeled Tf-NP-siRNA in NOD-SCID mice analyzed by confocal microscopy.

Figure 6. In vivo distribution study of Cy5 labeled Tf-NP-siRNA

(A) Tf-mediated tumor targeting distribution of Cy5-siRNA by IVIS imaging. 1–2: Cy5-labeled NP-siRNA in NOD-SCID mice. 3: Cy5-labeled NP-siRNA in normal SCID mice. 4–5: Cy5-labeled Tf-NP-siRNA in NOD-SCID mice. 3: Cy5-labeled Tf-NP-siRNA in normal SCID mice. (B) Cy5-labeled Tf-NP-siRNA in NOD-SCID mice analyzed by confocal microscopy.

Figure 7. In vivo down-regulation study of Tf-NP-LOR-1284 in tumor-bearing NOD-SCID mice

(A) R2 mRNA expression of tumors in different groups. (B). R2 protein expression of tumors in different groups.