Evaluation of intraductal delivery of poly(ethylene glycol)-doxorubicin conjugate nanocarriers for the treatment of ductal carcinoma in situ (DCIS)-like lesions in rats

Abstract

Ductal carcinoma in situ is the most commonly diagnosed early stage breast cancer. The efficacy of intraductally delivered poly(ethylene glycol)-doxorubicin (PEG-DOX) nanocarriers, composed of one or more DOX conjugated to various PEG polymers, was investigated in an orthotopic ductal carcinoma in situ-like rat model. In vitro cytotoxicity was evaluated against 13762 Mat B III cells using MTT assay. The orthotopic model was developed by inoculating cancer cells into mammary ducts of female Fischer 344 retired breeder rats. The ductal retention and in vivo antitumour efficacy of two of the six nanocarriers (5 kDa PEG-DOX and 40 kDa PEG-(DOX)₄) were investigated based on in vitro results. Mammary retention of DOX and PEG-DOX nanocarriers was quantified using in vivo imaging. Histopathologic effects of DOX and PEG-DOX nanocarriers on mammary ductal structure were also investigated. Cytotoxicities of small linear PEG-DOX nanocarriers (5 and 10 kDa) were not different from DOX whereas larger PEG-DOX nanocarriers showed reduced potency. The order of mammary retention was 40 kDa PEG-(DOX)₄ > 5 kDa PEG-DOX >> DOX, in normal and tumour-bearing rats. Intraductally administered PEG-DOX nanocarriers and DOX were effective in reducing tumour incidence and increasing survival rate, with no significant differences found among the three treatment groups. However, nanocarriers administered intravenously at the same doses were not effective, and intraductally administered free DOX caused severe local toxicity. Intraductal administration of PEG-DOX nanocarriers is effective and less toxic than that of free DOX, as well as IV DOX/PEG-DOX. Furthermore, PEG-DOX nanocarriers demonstrate the added benefit of prolonging DOX ductal retention, which would necessitate less frequent dosing.

Keywords: DCIS rat model; PEG nanocarrier; doxorubicin; ductal carcinoma in situ (DCIS); intraductal therapy; mammary gland retention.

Figure 1

Correlation between the reduction in the ratio (PEG‐DOX vs. DOX) of in vitro IC₅₀ against 13762 Mat B III from Table 1 versus linear PEG molecular weight in PEG‐DOX nanocarriers, R ² = 0.98. DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 2

Tumourigenicity of 13762 Mat B III cells inoculated intraductally in F344 rats. The rats were injected with 2.5 × 105 cells/duct and thereafter were killed at the indicated time points. (A) Representative microscopic images of mammary whole‐mounts. The images were acquired with 2× objective. (B) Hematoxylin and eosin staining of paraffin sections of corresponding mammary samples from F344 rats. Microscopic images were taken with a 40× objective.

Figure 3

Optical imaging of DOX and PEG‐DOX nanocarriers dosed intraductally into mammary glands of F344 rats. The samples were administered into the mammary ducts of the rats and then imaged at different time points using an In‐Vivo MS FX PRO^® optical imaging system. (A) F344 normal rats; (B) F344 rat tumour model at day 2 after tumour cell inoculation (n = 3). No difference in mammary retention was observed between (A) normal and (B) tumour‐bearing rats (Fig. 4 and Table 2). DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 4

Mammary retention of DOX and PEG‐DOX nanocarriers in F344 rats. DOX and PEG‐DOX nanocarriers were administered intraductally into (A) normal and (B) tumour‐bearing rats. Fluorescence intensities were measured with an optical imager over time, as shown in Figure 3. Each point represents mean ± standard deviation (n = 3). DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 5

Correlation between poly(ethylene glycol)‐doxorubicin nanocarrier molecular weight and mammary retention half‐life. R ² = 0.95 and 1.00 in normal and tumour‐bearing rats, respectively.

Figure 6

Effect of PEG‐DOX nanocarriers administered by different routes on tumour growth in orthotopic breast cancer model. F344 retired female breeder rats were inoculated with 13762 Mat B III cells (2.5 × 105 cells/rat) into the fourth mammary duct. Two days later, the rats received either intraductal (ID) treatment into the same duct or intravenous (IV) treatment with DOX or PEG‐DOX nanocarriers at doses of 0.83 mg/kg DOX equivalents. (A) Control group, rats received no treatment; (B) rats received intraductal treatment; (C) rats received intravenous treatment. Each point represents the mean ± standard deviation (n = 3–9). DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 7

Intraductal treatment with DOX and PEG‐DOX nanocarriers increased survival in the orthotopic breast cancer model. (A) Rats treated with intraductal therapy (ID); (B) rats treated with intravenous therapy (IV). There is insignificant difference among all ID‐treated groups (A). DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 8

Magnetic resonance imaging images of F344 rat mammary glands treated with DOX and PEG‐DOX nanocarriers. The rats were treated with 0.83 mg/kg/duct of DOX and were scanned for magnetic resonance imaging images after 65 days. Soft lumps were observed in mammary glands (arrowhead) of DOX‐treated rats but not in those receiving PEG‐DOX nanocarriers. The soft lumps were considered to be due to local toxicity of intraductal administration of free DOX. DOX, doxorubicin; PEG, poly(ethylene glycol).

Figure 9

Histology of F344 rat mammary ducts treated with DOX and PEG‐DOX nanocarriers. DOX or PEG‐DOX nanocarriers were intraductally administered into normal and tumour‐bearing rats. The animals were euthanized at 65 days posttreatment. Paraffin sections were stained with hematoxylin and eosin. (A) Normal rats treated with DOX or PEG‐DOX nanocarriers; (B) tumour‐bearing rats treated with DOX or PEG‐DOX nanocarriers. Arrows indicate morphological changes in the sections. Microscopic images were taken at 40× objective. DOX, doxorubicin; PEG, poly(ethylene glycol).