Comparison of active and passive targeting of doxorubicin for somatostatin receptor 2 positive tumor models by octreotide-modified HPMA copolymer-doxorubicin conjugates

Figures & data

Figure 1. Synthesis of non-modified and Oct-modified HPMA copolymer conjugates with Dox.

Table 1. Characteristics of the synthesized HPMA copolymers.

HPMA copolymers	Mw (kDa)	PDI	Mol% Peptide	wt% DOX	mol% FITC
P-DOX	28.3	1.64	−	5.18	−
P-DOX-Oct	33.1	1.82	3.9	5.76	−
P-FITC	16.9	1.42	−	−	1.7
P-FITC-Oct	22.8	1.50	2.0	−	1.7
^131I-P	20.7	1.45	−	−	−
^131I-P-Oct	22.3	1.47	3.3	−	−

Figure 2. Quantitative PCR analysis of expression levels of SSTR2 in HepG2, A549 and H₂₂ cells.

Figure 3. Internalization of P-FITC and P-FITC-Oct (equivalent to 50 mM of FITC) co-incubated with or without free Oct (0.04 mg/ml) in HepG2 (A–D) and A549 (E–H) cells by inverted fluorescence microscopy. The green signal indicated FITC-labeled conjugates while blue signal indicated nuclei. A: HepG2 cells incubated with P-FITC; B: HepG2 cells incubated with P-FITC and free Oct; C: HepG2 cells incubated with P-FITC-Oct; D: HepG2 cells incubated with P-FITC-Oct and free Oct; E: A549 cells incubated with P-FITC; F: A549 cells co-incubated with P-FITC and free Oct; G: A549 cells co-incubated with P-FITC-Oct; H: A549 cells incubated with P-FITC-Oct and free Oct.

Figure 4. Quantitative study of uptake of P-DOX and P-DOX-Oct in HepG2 and A549 cells in presence or absence of free Oct. A: cells were incubated with various concentrations of conjugates for 2 h. B: cells were incubated with conjugates (0.6 mg polymer/ml) and 0.04 mg/ml of free Oct (#p < 0.05 for P-DOX-Oct versus P-DOX-Oct with free Oct).

Figure 5. Morphological and nuclear changes of HepG2 (A-C) and A549 (D-F) cells treated with P-DOX and P-DOX-Oct for 24 h as determined by DAPI staining. A and D: control groups. B and E: P-DOX treated groups. C and F: P-DOX-Oct treated groups. The white arrows pointed to the apoptosis and necrosis.

Table 2. The IC₅₀ values of DOX, P-DOX and P-DOX-Oct against HepG2 and A549 cells (n = 3).

IC50(μg/ml Dox equiv.)	DOX	P-DOX	P-DOX-Oct
HepG2	0.014 ± 0.002	42.77 ± 2.95	25.43 ± 1.73*
A549	0.025 ± 0.003	59.50 ± 1.48	37.20 ± 2.46*

*p < 0.05 versus P-Dox.

Figure 6. In vivo biodistribution of non-modified (A) and Oct-modified (B) HPMA copolymers at 24, 72 and 120 h.

Table 3. The tumor-to-blood ratios of the ¹³¹I-labeled copolymers (n = 4).

	Tumor-to-blood ratio
Time(h)	^131I-P	^131I-P-Oct
24	0.318	0.661*
48	0.378	0.971#
72	0.436	1.366#

#p < 0.05, *p < 0.01 for ¹³¹I-P-Oct versus ¹³¹I-P.

Figure 7. Antitumor activity of DOX·HCl, P-DOX and P-DOX-Oct on Kunming mice bearing H₂₂ xenografts. The controls were administrated with saline. Tumor volume were recorded (n = 5, #p < 0.05, versus DOX·HCl) and tumors were harvested at the end of experiments (21 d).

Table 4. The mean tumor weight after treatments (n = 5).

Treatment	Mean tumor weight (g)	Inhibition ratio (%)
Saline	1.654 ± 0.358	−
DOX·HCl	0.982 ± 0.406	40.62
P-DOX	0.952 ± 0.343#	42.42
P-DOX-Oct	0.531 ± 0.334*	67.90

#p < 0.05, *p < 0.01, versus saline.