HPMA polymer-based site-specific delivery of oligonucleotides to hepatic stellate cells

Abstract

The objective was to determine whether bioconjugation of type I collagen specific triplex forming oligonucleotide (TFO) to N-(2-hydroxypropyl) methacrylamide (HPMA) containing tetrapeptide Gly-Phe-Leu-Gly (GFLG) and mannose 6-phosphate (M6P) can provide their targeted delivery to hepatic stellate cells (HSCs). Following bioconjugation, M6P-GFLG-HPMA-GFLG-32P-TFO was characterized by PAGE, HPLC, and GPC, and then its biodistribution was determined. TFO was dissociated from the conjugate when incubated with papain and formed triplex with the target DNA duplex. Type 1 collagen gene expression was significantly inhibited when HSC-T6 cells were transfected with this conjugate. Following tail vein injection into rats, M6P-GFLG-HPMA-GFLG-(32)P-TFO was rapidly cleared from the circulation and accumulated mainly in the liver. The plasma concentration versus time profile was biphasic, with 12.37 min as t(1/2) of distribution and 2886.48 min as t(1/2) of elimination. A large proportion of the injected M6P-GFLG-HPMA-GFLG-32P-TFO was taken up by the HSCs of both normal and fibrotic rats, which were isolated by liver perfusion at 30 min post-injection. Preinjection of M6P-GFLG-HPMA-GFLG-ONP into fibrotic rats decreased the liver uptake of the conjugates from 60% to 13%, suggesting M6P/TGFII receptor-mediated endocytosis of the conjugates by HSCs. Almost 80% of the total liver uptake in fibrotic rats was contributed by HSCs. In conclusion, conjugation with M6P-HPMA-GFLG significantly increased TFO delivery to the HSCs and could be potentially used for treating liver fibrosis.

Figure 1

Synthesis scheme of M6P-GFLG-HPMA-GFLG-TFO.

Figure 2

HPLC chromatography of M6P-GFLG-HPMA-GFLG-TFO conjugate. There is a new peak for conjugate different from the reactants, such as M6P, poly(HPMA-co-GFLG-ONP) and TFO.

Figure 3

Polyacrylamide gel electrophoresis (PAGE) of M6P-GFLG-HPMA-GFLG-TFO. A) methylene blue staining. B) autoradiography. There was no band shift for the conjugate.

Figure 4

TFO dissociation from M6P-GFLG-HPMA-GFLG-TFO by papain. The conjugate was treated with papain for 1h, 2h, 8h, 24h followed by polyacrylamide gel electrophoresis (PAGE) and autoradiography. The conjugate not treated with papain was used as a control.

Figure 5

Triplex formation with target duplex DNA after release from M6P-GFLG-HPMA-GFLG-³²P-TFO conjugate. Samples were applied on 15% native PAGE at 4 °C in 89 mM Tris-borate buffer, containing MgCl₂ (20 mM) for 4 h. Lane 1: ³²P-TFO; lane 2: duplex DNA/³²P-TFO (200:1) incubated for 24 h; lane 3: Duplex DNA/³²P-TFO (200:1) and papain incubated for 24 h; lane 4: M6P-GFLG-HPMA-GFLG-³²P-TFO; lane 5: M6P-GFLG-HPMA-GFLG-³²P-TFO incubated with papain for 24 h at 37 °C; and lane 6: duplex DNA/M6P-GFLG-HPMA-GFLG-³²P-TFO (200:1) incubated for 24 h at 37 °C with papain.

Figure 6

Inhibition of collagen gene expression. Transfection of HSC-T6 cells with TFO (lane 3) and M6P-GFLG-HPMA-GFLG-TFO (lane 2) inhibited collagen gene expression compared to the control group (lane 1). Western blot analysis of collagen primary antibody provided Santa Cruz was also shown in the right figure.

Figure 7

Biodistribution of M6P-GFLG-HPMA-GFLG-³²P-TFO and free ³²P-TFO after tail vein injection into rats at a dose of 0.2 mg TFO/kg of body weight.

Figure 8

Concentration time profiles of radioactivity in different organs and the plasma after tail vein injection of M6P-GFLG-HPMA-GFLG-³²P-TFO and free ³²P-TFO into rats at a dose of 0.2 mg TFO/kg of body weight.

Figure 9

Pharmacokinetic profiles of M6P-GFLG-HPMA-GFLG-³²P-TFO at 30min after post vein injection. Plasma data was analyzed using a two-compartment model with WinNonlin Enterprise (version 5.2) software. AUC = Area under the curve; CL = Clearance; V_d = Volume of distribution.

Figure 10

Effect of fibrosis on hepatic uptake of M6P-GFLG-HPMA-GFLG-³²P-TFO and HPMA-³²P-TFO after systemic administration into DMN induced fibrotic rats. At 30 min postinjection of this conjugate (1×10⁶cpm) at a dose of 0.2mg/kg, blood was collected by cardiac puncture. Rats were sacrificed; major organ were isolated, washed with saline and subjected into scintillation counter. Values are the mean ± S.D. of 3 rats.

Figure 11

Effect of excess M6P-GFLG-HPMA on the biodistribution of M6P-GFLG-HPMA-GFLG-³²P-TFO in fibrotic rats.

Figure 12

Intrahepatic distribution of M6P-GFLG-HPMA-GFLG-³²P-TFO in normal and fibrotic rats. The liver was perfused in situ by collagenase/pronase digestion at 30 min post-injection of M6P-GFLG-HPMA-GFLG-³²P-TFO, HPMA-GFLG-³²P-TFO or ³²P-TFO at dose of 0.2 mg TFO/kg of body weight. Hepatocytes, Kupffer and endothelial cells, and hepatic stellate cells (HSC) were separated and the associated radioactivity was measured. The contribution of each liver cell type was exposed as percentage of total liver uptake. Results are expressed as the mean ± S.D. (n=3).