In vitro and in vivo evaluation of tumor targeting styrene-maleic acid copolymer-pirarubicin micelles: Survival improvement and inhibition of liver metastases - PubMed (original) (raw)

In vitro and in vivo evaluation of tumor targeting styrene-maleic acid copolymer-pirarubicin micelles: Survival improvement and inhibition of liver metastases

Jurstine Daruwalla et al. Cancer Sci. 2010 Aug.

Abstract

Pirarubicin is a derivative of doxorubicin with improved intracellular uptake and reduced cardiotoxicity. We have prepared a micellar formulation of pirarubicin using styrene-maleic acid copolymer (SMA) of mean molecular weight of 1.2 kDa, which exhibits a mean diameter of 248 nm in solution. Being a macromolecule, SMA-pirarubicin micelles exhibit excellent tumor targeting capacity due to the enhanced permeability and retention (EPR) effect. Here we report the antitumor activity of SMA-pirarubicin micelles on human colon and breast cancer cell lines in vitro, and a murine liver metastasis model in vivo. Metastatic tumor microvasculature, necrosis, apoptosis, proliferation, and survival were also investigated using immunohistochemistry for Ki-67, active caspase-3, and CD34, respectively. Drug cytotoxicity in vitro was assessed using MTT (3-[4,5-dimethyl-2-thiazolyl]-2, 5-diphenyl-2H-tetrazolium bromide) assay. In vivo, SMA-pirarubicin was administered at 100, 150, or 200 mg/kg (pirarubicin equivalent). Tumor microvasculature was also assessed using scanning electron microscopy. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles were toxic against human colorectal and breast cancer cells in vitro. IC(50) was at or below 1 muM, free pirarubicin equivalent. In vivo, SMA-pirarubicin at 100 mg/kg reduced tumor volume by 80% and achieved a survival rate of 93% at 40 days after tumor inoculation. Styrene-maleic acid copolymer (SMA)-pirarubicin micelles demonstrated potent antitumor activity in this liver metastases model, contributing to prolonged survival. Histological examination of tumor nodules showed significant reduction and proliferation of tumor cells (>90%). The present results suggest that investigation of the effect of multiple dosing at later time points to further improve survival is warranted.

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Figures

Figure 1

Representation of the chemical structures. (a) Styrene‐maleic acid copolymer (SMA) (n = 4–14). (b) Anticancer agent pirarubicin. (c) Putative structure of SMA‐pirarubicin micelles. Brackets indicate copolymer of SMA.

Figure 2

Cytotoxic effect of styrene‐maleic acid copolymer (SMA)‐pirarubicin on tumor cell lines (a) MoCR mouse colon, (b) DLD‐1 human colon, (c) HT‐29 human colon, (d) MCF‐7 human breast adenocarcinoma investigated at 0.3, 1, 3, and 10 μM. Cell viability was measured using MTT (3‐[4,5‐dimethyl‐2‐thiazolyl]‐2, 5‐diphenyl‐2H‐tetrazolium bromide) assay. All cell lines were sensitive to SMA‐pirarubicin in a dose‐dependent manner. The IC50c for SMA‐pirarubicin for all cell lines was 1 μM ± 0.1. MoCR and HT‐29 cells were more sensitive compared to DLD‐1 and MCF‐7 cells. (*P < 0.005, **P < 0.001).

Figure 3

Macroscopic images of livers with tumors. Styrene‐maleic acid copolymer (SMA)‐pirarubicin was administered at 100, 150, or 200 mg/kg (i.v.) in three divided doses (14, 16, and 18 days after tumor implantation). Control received saline intra‐venously. Styrene‐maleic acid copolymer (SMA)‐pirarubicin produced a marked reduction in liver metastases compared to untreated livers, with significant reduction in the number and size of tumor nodules.

Figure 4

(a) Quantitative stereology was used to calculate tumor volume and number of liver metastases. Livers were sectioned (1.5 mm) and area of tumor and normal liver was traced using image analysis software. Styrene‐maleic acid copolymer (SMA)‐pirarubicin micelles (100 mg/kg) reduced tumor volume to 10% of the control. (b) Styrene‐maleic acid copolymer (SMA)‐pirarubicin micelles reduced number of liver metastases by more than 80% (**P < 0.001,

anova

with Tukey method). Higher doses were of no further benefit. The number of tumor nodules in the treated groups was reduced to 50% of the control.

Figure 5

Survival advantage of styrene‐maleic acid copolymer (SMA)‐pirarubicin micelles was assessed. Dose of 33 mg/kg (equivalent to free pirarubicin) was given three times on day 14, 16, and 18, a total dose of 100 mg/kg. On day 40 median survival was 97% in the SMA‐pirarubicin treated group, compared to 7% survival in the control group (P < 0.001). Mortality without treatment was 100% by day 44, whereas 40% survival was observed on day 60 with drug treatment.

Figure 6

Livers at day 21 (72 h after styrene‐maleic acid copolymer (SMA)‐pirarubicin injection) were excised and fixed in formalin for histological analysis. The percentage of tumor necrosis was quantified on H&E‐stained paraffin sections. (a) SMA‐pirarubicin (100 mg/kg) resulted in 3‐fold increase in tumor necrosis (**P < 0.001). (b) Control tumors demonstrated poor‐to‐moderate differ‐entiation. Tumors were cohesive (T) with minimal central necrosis and displaced the surrounding normal liver (NL). (c) Drug treated tumors were extensively damaged; high degree of central necrosis extending to the periphery (black arrows). (c–e) Treated tumors were surrounded by a fibrous capsule which was absent in untreated tumors. (d) Insert, fibrous capsule. Treated tumor cells exhibited hydropic swelling and enlarged nuclei. (f) Scanning electron micrograph of untreated tumor demonstrating dilated tumor vessels (TV). (g) Following treatment, the central tumor core is void of vessels, and appears necrotic (N). Vessels from the periphery have become occluded resulting in tapered ends (white arrows) indicative of vascular shut down – a potential mechanism of tumor destruction. Scale bars, (a–c) 200 μm, (d) 100 μm.

Figure 7

Apoptotic index was calculated as a percentage of the total number of cells using immunohistochemistry for active caspase‐3 and H&E morphological criteria. (>10 000 cells counted treatment group). Morphologically apoptotic cells on H&E staining demonstrated a halo surrounding the condensed nuclei (c, white arrows). Apoptotic cells were demarcated by intense brown staining (DAB method) (e, black arrows). Drug treatment (c,e) significantly elevated apoptosis compared to control (b,d). Scale bars, 50 μm.

Figure 8

Ki‐67 immunohistochemistry was used to assess tumor proliferation. Parts (b) and (c) are no drug control; (d), (e), and (f) are drug‐treated groups. Proliferating cells were demarcated by intense brown DAB staining (e, H&E staining). (a) Styrene‐maleic acid copolymer (SMA)‐pirarubicin reduced tumor proliferation by 30–40%, 72 h following the final drug dose, whereas the drug treated cells shows significant necrosis (d). In (b,c), control tumors demonstrated a high degree of tumor proliferation. In (d), tumor proliferation was restricted to a thick viable band at the periphery of tumors with extensive central necrosis. In (e), proliferation in SMA‐pirarubicin treated tumors was restricted to the thin viable rim at the tumor periphery (cf. d,f). The degree of proliferation in the viable rim of control tumors (b,c) is denser than that of drug‐treated tumors (d,f). The fibrous capsule surrounding treated tumors seldom contained proliferating tumor cells (e,f, black arrows). Scale bars, 200 μm.

Figure 9

Effect of styrene‐maleic acid copolymer (SMA)‐pirarubicin on tumor vessels. (a,b) Control, (c,d) drug treated liver section. (a,c) Immunohistochemistry for CD34. Control tumor vessels demonstrated a high degree of CD34 positive staining (a). Vessels were visibly patent and dispersed throughout the tumor and in close proximity to each other (shown by arrows). In drug‐treated liver, most surviving vessels were localized at the tumor–host interface (d) and were concentrically arranged around the tumor (c). Scanning electron microscopy images show drug treatment resulted in complete vascular disappearance centrally (d), consistent with CD34 and H&E staining. (V) denotes viable subcapsular tumor.

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In vitro and in vivo evaluation of tumor targeting styrene-maleic acid copolymer-pirarubicin micelles: Survival improvement and inhibition of liver metastases - PubMed (original) (raw)