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. 2010 Jan 4;141(1):62-9.
doi: 10.1016/j.jconrel.2009.08.019. Epub 2009 Aug 28.

Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing

Chee Guan Koh  1 , Xulang Zhang, Shujun Liu, Sharon Golan, Bo Yu, Xiaojuan Yang, Jingjiao Guan, Yan Jin, Yeshayahu Talmon, Natarajan Muthusamy, Kenneth K Chan, John C Byrd, Robert J Lee, Guido Marcucci, L James Lee
Affiliations

Delivery of antisense oligodeoxyribonucleotide lipopolyplex nanoparticles assembled by microfluidic hydrodynamic focusing

Chee Guan Koh et al. J Control Release. .

Abstract

A multi-inlet microfluidic hydrodynamic focusing (MF) system to prepare lipopolyplex (LP) containing Bcl-2 antisense deoxyoligonucleotide (ODN) was developed and evaluated. The lipopolyplex nanoparticles consist of ODN:protamine:lipids (1:0.3:12.5wt/wt ratio) and the lipids included DC-Chol:egg PC:PEG-DSPE (40:58:2mol/mol%). Using K562 human erythroleukemia cells, which contain an abundance of Bcl-2 and overexpression of transferrin receptors (TfR), and G3139 (oblimerson sodium or Genasense(TM)) as a model cell line and drug, respectively, the Bcl-2 down-regulation at the mRNA and protein levels as well as cellular uptake and apoptosis was compared between the conventional bulk mixing (BM) method and the MF method. The lipopolyplex size and surface charge were characterized by dynamic light scattering (DLS) and zeta potential (zeta) measurement, respectively, while the ODN encapsulation efficiency was determined by gel electrophoresis. Cryogenic transmission electron microscopy (Cryo-TEM) was used to determine the morphology of LPs. Our results demonstrated that MF produced LP nanoparticles had similar structures but smaller size and size distribution compared to BM LP nanoparticles. MF LP nanoparticles had higher level of Bcl-2 antisense uptake and showed more efficient down-regulation of Bcl-2 protein level than BM LP nanoparticles.

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Figures

Fig. 1
Fig. 1
BM preparation of ODN-LP (A) and (B) transferrin conjugated PEG–DSPE (Tf– PEG–DSPE). (A) Step 1: after mixing ODN with protamine/lipids and before dialysis, 2: after dual dialysis, 3: after 0.2 μm filtering, and 4: after post insertion with Tf–PEG– DSPE. (B) Holo-transferrin is reacted with Traut's reagent to from thiolated transferrin (Holo-Tf-SH) and reacted with maleimide–DSPE–PEG to form Tf–PEG–DSPE micelles for post insertion.
Fig. 2
Fig. 2
A 5-inlet MF device. (A) Schematic of the 5-inlet MF system. (B) Optical micrograph of the flow pattern at the two junctions (X and Y) of the MF system. (C) Fluorescence micrograph of flow pattern at junction Y. The volumetric flow rates used for rhodamine, fluorescein, and rhodamine were 200, 20, and 200 μL/min, respectively. Red and green color is rhodamine and fluorescein, respectively. Scale bar=250 μm.
Fig. 3
Fig. 3
Cryo-TEM images of LP nanoparticles prepared by (A) BM and (B) MF. (A) Arrowheads show multilamellar vesicles, while arrows show onion-like complexes. (B) Onion-like aggregates (white arrow), amorphous protamine/ODN aggregate (black arrow), and multilamellar nanostructure in which protamine/ODN aggregate coats lipid bilayers (arrowhead). Scale bars represent 100 nm.
Fig. 4
Fig. 4
Determination of ODN encapsulation efficiency in LP nanoparticles by agarose gel electrophoresis. LPs after dialysis were lysed in 1% sodium dodecyl sulfate (SDS) at 95 °C for 5 min. Lanes 1–3. ODN; 4. BM LP without 1% SDS; 5–6. BM LP with 1% SDS; 7. MF LP without 1% SDS; 8–9. MF LP with 1% SDS.
Fig. 5
Fig. 5
Uptake of BM and MF lipopolyplexes containing FITC-labeled ODN in K562 cells. Cells were treated with non-targeted and targeted BM and MF LP nanoparticles staining FITC-labeled ODN as analyzed by (A) flow cytometry and (B) fluorescence microscopy at 400× magnification. 1 is untreated cell control, 2 is cells treated with targeted BM Tf-LP, and 3 is cells treated with targeted MF Tf-LP. (C) Mean fluorescence intensity of cells treated with MF Tf-LP and BM Tf-LP nanoparticles. Cells were treated for 24 h. The ODN concentration used was 0.5 μM at a cell density of 3×105. (n=3).
Fig. 6
Fig. 6
Effect of Bcl-2 down-regulation by ODN. K562 cells were treated with free ODN, BM Tf-LP, MF LP and MF Tf-LP. ODN concentration was 1 μM in all groups except for the untreated group. A representative Western blot of Bcl-2 protein expression (A), its corresponding densitometry data (B), and results of real-time RT-PCR analysis (C) at 24 and 48 h following treatment are shown. p<0.05 and p<0.01 indicated by * and ** symbols, respectively, as compared to MF Tf-LP sample (n=3). □ Untreated control; formula imageFree ODN; formula imageBM Tf-LP, 1.0 μM ODN; formula imageMF LP, 1.0 μM ODN; and formula imageMF Tf-LP, 1.0 μM ODN.
Fig. 7
Fig. 7
Effect of ODN concentration on Bcl-2 down-regulation. A representative Western blot of Bcl-2 protein expression (A) and its corresponding densitometry data (B) at 24 and 48 h following treatment with free ODN and ODN-containing formulations are shown. K562 cells were treated with BM Tf-LP and MF Tf-LP at ODN concentration of either 0.5 μM or 1.0 μM. p<0.05 and p<0.01 indicated by * and ** symbols, respectively, as compared to MF Tf-LP sample (n=3). For free ODN, 1.0 μM was used. □ Untreated control; formula imageFree ODN, 1.0 μM ODN; formula imageBM Tf-LP, 0.5 μM ODN; formula imageMF Tf-LP, 0.5 μM ODN; formula imageBM Tf-LP, 1.0 μM ODN; and formula imageMF Tf-LP, 1.0 μM ODN.
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