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PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma

View Article: PubMed Central - PubMed

ABSTRACT

Hepatic carcinoma (HCC) is a lethal disease associated with high morbidity and poor prognosis. Recently years, gene therapies have offered novel modalities to improve the prognosis of HCC patients. MicroRNA-99a (miR-99a) is frequently down-regulated in HCC, where it acts as a tumor suppressor. Therefore, we constructed monomethoxy (polyethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine)-lactobionic acid- anti-vascular endothelial growth factor antibody (mPEG-PLGA-PLL-LA/VEGFab or PEAL-LA/VEGFab) nanoparticles (NPs) with highly specific targeting properties as carriers to restore the expression of miR-99a both in vitro and in vivo, to inhibit HCC progression. In vitro, PEAL-LA/VEGFab NPs showed more efficient delivery of miR-99a to HepG2 cells than the conventional transfection reagent LipofectamineTM2000 (Lip2000). The higher delivery efficiency associated with PEAL-LA/VEGFab NPs consequently resulted in down-regulation of target genes and suppression of the proliferation, migration and invasion of HepG2 cells. In vivo, miR-99a-PEAL-LA/VEGFab NPs inhibited tumor xenograft growth in HCC-bearing mice without causing obvious systemic toxicity. Our results demonstrate that PEAL-LA/VEGFab NPs selectively and effectively deliver miR-99a to HCC cells based on the double-targeting character of these nanoparticles, thereby offering potential for translation into effective clinical therapies for HCC.

No MeSH data available.


(A) CLSM images of HepG2 cells incubated with Cy5(red)-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 h. Magnification 200 ×, the scale bar is 100 μm. (B) Flow cytometry analysis of HepG2 cells treated with Cy5-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 hours.
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f3: (A) CLSM images of HepG2 cells incubated with Cy5(red)-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 h. Magnification 200 ×, the scale bar is 100 μm. (B) Flow cytometry analysis of HepG2 cells treated with Cy5-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 hours.

Mentions: CLSM analysis was conducted to evaluate the delivery efficiency of miR-99a by NPs. To facilitate observations by CLSM, miR-99a was labeled with the Cy5 fluorescent probe to form Cy5-miR-99a-PEAL-LA NPs and Cy5-miR-99a-PEAL-LA/VEGFab NPs. The commercial transfection reagent LipofectamineTM2000 (Lip2000) was used as a control. As shown in Fig. 3A, after 4 h of co-incubation, the fluorescent signal (red) in cells treated with Cy5-miR-99a-PEAL-LA NPs was similar to those incubated with Cy5-miR-Lip2000, suggesting that PEAL-LA NPs can achieve similar transfection efficiencies to Lip2000. Furthermore, cells cultured with Cy5-miR-99a-PEAL-LA/VEGFab NPs exhibited much brighter fluorescence emission than those cultured with Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-Lip2000, suggesting that Cy5-miR-99a-PEAL-LA/VEGFab NPs containing dual-targeting moieties (LA and VEGFab) exhibit a synergistic targeted effect and show significant enhancement of miR-99a delivery.


PLGA-based dual targeted nanoparticles enhance miRNA transfection efficiency in hepatic carcinoma
(A) CLSM images of HepG2 cells incubated with Cy5(red)-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 h. Magnification 200 ×, the scale bar is 100 μm. (B) Flow cytometry analysis of HepG2 cells treated with Cy5-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 hours.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5384185&req=5

f3: (A) CLSM images of HepG2 cells incubated with Cy5(red)-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 h. Magnification 200 ×, the scale bar is 100 μm. (B) Flow cytometry analysis of HepG2 cells treated with Cy5-miR-99a-Lip2000, Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-PEAL-LA/VEGFab NPs for 4 hours.
Mentions: CLSM analysis was conducted to evaluate the delivery efficiency of miR-99a by NPs. To facilitate observations by CLSM, miR-99a was labeled with the Cy5 fluorescent probe to form Cy5-miR-99a-PEAL-LA NPs and Cy5-miR-99a-PEAL-LA/VEGFab NPs. The commercial transfection reagent LipofectamineTM2000 (Lip2000) was used as a control. As shown in Fig. 3A, after 4 h of co-incubation, the fluorescent signal (red) in cells treated with Cy5-miR-99a-PEAL-LA NPs was similar to those incubated with Cy5-miR-Lip2000, suggesting that PEAL-LA NPs can achieve similar transfection efficiencies to Lip2000. Furthermore, cells cultured with Cy5-miR-99a-PEAL-LA/VEGFab NPs exhibited much brighter fluorescence emission than those cultured with Cy5-miR-99a-PEAL-LA NPs or Cy5-miR-99a-Lip2000, suggesting that Cy5-miR-99a-PEAL-LA/VEGFab NPs containing dual-targeting moieties (LA and VEGFab) exhibit a synergistic targeted effect and show significant enhancement of miR-99a delivery.

View Article: PubMed Central - PubMed

ABSTRACT

Hepatic carcinoma (HCC) is a lethal disease associated with high morbidity and poor prognosis. Recently years, gene therapies have offered novel modalities to improve the prognosis of HCC patients. MicroRNA-99a (miR-99a) is frequently down-regulated in HCC, where it acts as a tumor suppressor. Therefore, we constructed monomethoxy (polyethylene glycol)-poly(D,L-lactide-co-glycolide)-poly(L-lysine)-lactobionic acid- anti-vascular endothelial growth factor antibody (mPEG-PLGA-PLL-LA/VEGFab or PEAL-LA/VEGFab) nanoparticles (NPs) with highly specific targeting properties as carriers to restore the expression of miR-99a both in vitro and in vivo, to inhibit HCC progression. In vitro, PEAL-LA/VEGFab NPs showed more efficient delivery of miR-99a to HepG2 cells than the conventional transfection reagent LipofectamineTM2000 (Lip2000). The higher delivery efficiency associated with PEAL-LA/VEGFab NPs consequently resulted in down-regulation of target genes and suppression of the proliferation, migration and invasion of HepG2 cells. In vivo, miR-99a-PEAL-LA/VEGFab NPs inhibited tumor xenograft growth in HCC-bearing mice without causing obvious systemic toxicity. Our results demonstrate that PEAL-LA/VEGFab NPs selectively and effectively deliver miR-99a to HCC cells based on the double-targeting character of these nanoparticles, thereby offering potential for translation into effective clinical therapies for HCC.

No MeSH data available.