Nanometronomic treatment of 4T1 breast cancer with nanocaged doxorubicin prevents drug resistance and circumvents cardiotoxicity

Figures

• 

  Figure 1. In vitro activity of HFn-DOX nanocages toward tumor cells a. Intracellular localization of HFn nanoparticles. Confocal microscopy merged images of 4T1-L cells, incubated for 15 min, 1, 3 and 48 h at 37 °C with 100 μg mL−1 of FITC-labeled HFn (green). Nuclei were stained with DAPI (blue). Scale bar: 10 μm. b. HFn binding toward 4T1-L breast cancer cells. 4T1-L cells were incubated 2 h at 4 °C with FITC-labeled HFn (20 and 100 μg mL−1) and then processed for flow cytometry. Untreated cells were used as control to set the positive region. c. Proliferation profiles of cells treated with 1 μM DOX or HFn-DOX for up to 72 h. Untreated cells are used as control. Values are mean of six replicates ± SE. d. Viability of cells treated with free or nanoformulated DOX. 4T1-L cells were treated with 1, 0.1, and 0.01 μM DOX or HFn-DOX for up to 72 h. Viability was assessed by measuring the conversion of MTT into formazan, normalized on cell proliferation of untreated cells. Statistical significance vs. CTRL #P<0.05, ##P<0.005; Statistical significance vs. DOX *P<0.01; **P<0.005. e. Cell death assay using DOX or HFn-DOX. 4T1-L cells were treated with 1, 0.1, and 0.01 μM DOX or HFn-DOX for 3 or 24…

• 

  Figure 2. In vivo targeting and distribution of void HFn nanocarrier a. Epifluorescence (Epf) images of mice bearing 4T1-L tumors acquired 1, 2, 24 and 48 h after intravenous (i.v.) injection into the tail vein of 5 μg kg−1 AF660-HFn and b. averaged Epf intensity of the bladder region of interest (ROI). c. Epf of isolated 4T1 tumors and averaged Epf intensity of tumor ROI acquired 1, 2, 24 and 48 h after exposure to HFn. d. Epf of isolated spleen (S), kidneys (K), liver (L), brain (B), heart (H), lungs (Lu), and e. averaged Epf intensity of the ROI obtained after 1, 2, 24 and 48 h exposure to HFn. f. Fluorescence intensity of urine collected 1, 2, 24 and 48 h after i.v. injection of AF660-HFn. The color scale in panels a, c and d indicates the averaged epifluorescence expressed as radiant efficiency [(p/sec/cm2/sr)/(mW/cm2)], where p/sec/cm2/sr is the number of photons per second that leave a square centimeter of tissue and radiate into a solid angle of one steradian (sr). Values reported in panels b, c, e and f are mean ± SE of at least 4 different samples under each experimental condition.

• 

  Figure 3. Bioavailability and tumor accumulation of HFn-DOX in comparison with free DOX a. Bioavailability of DOX and HFn-DOX at different time points. Plasma concentration of DOX after i.v. injection of HFn-DOX (black circles) or DOX (gray triangles) at 1.24 mg kg−1 and 12.4 mg kg−1 in healthy mice. b. DOX accumulation at 4T1-L tumor in mice at different time points after administration of 1.24 mg kg−1 DOX as free molecule or HFn-DOX. Female Balb/C mice orthotopically implanted with 4T1-L murine mammary carcinoma cells were injected 6 days after implantation (time 0) with DOX or HFn-DOX. DOX levels in tumor have been determined 1, 2, 4, and 24 h after i.v. injection following acidified isopropanol extraction from tumor homogenates. Aliquots from six mice per each time point concentration have been extracted and analyzed by spectrofluorimeter. Reported values are means of 3 samples/group ± SE. P values are summarized in Table S5. c. Confocal microscopy images of 4T1-L cells dissociated from tumor harvested 2 h after i.v. injection of DOX and HFn-DOX. DOX signal is represented in green, while nuclei were stained with DAPI (blue). Scale bar: 10 μm.

• 

  Figure 4. Efficacy of LDNM treatment with HFn-DOX a. DOX and HFn-DOX in vivo efficacy. Bioluminescence imaging of female Balb/C mice (n = 12/group) orthotopically implanted (day 0) with 4T1-L murine mammary carcinoma cells were treated with placebo or with 1.24 mg kg−1 of DOX, pl-DOX or HFn-DOX. Drug injections were performed into the tail vein at day 5, 9, 13 and 17. Mice were sacrificed at day 21. b. Quantification of tumor volume. Tumor volume was quantified by measuring the bioluminescence intensity signal of 4T1-L cells 5 min after intraperitoneal injection of luciferin. Dots represent the normalized mean value of BLI tumor signal ± SE. Statistical significance vs. placebo #P<0.05 ###P<0.005; vs. DOX *P<0.05 ***P<0.005. c. Quantification of apoptosis in tumor tissue upon treatment with HFn-DOX. Tumors excised at day 21 (n = 6/group) were fixed with formalin and embedded in paraffin. Histological slides were processed to label DNA fragments of apoptotic cells. Reported values are the mean of apoptotic cells number/field/sample ± SE. The count was performed on 10 fields/sample. Magnification 20×. Statistical significance vs. placebo ###P<0.005; vs. DOX ***P<0.005.

• 

  Figure 5. Impact of LDNM regimen on tumor angiogenesis and chemoresistance a. Quantification of angiogenesis in tumor tissue upon treatment with HFn-DOX. Tumors excised at day 21 (n = 5/group) were fixed with formalin and embedded in paraffin. Immunohistochemistry of histological slides were processed to label CD31+ cells. Reported values are mean of vessel number counted in 10 fields/sample ± SE. Magnification 40×. Statistical significance vs. placebo #P<0.005 ###P<0.00005; vs. DOX ***P<0.00005; vs. pl-DOX §§§P<0.0005. b. Quantification of MDR-1-expression. Excised tumors (n = 5/group) were processed for immunohistochemistry of MDR-1 antigen. The percentage of image area positive for MDR-1 expression was quantified using ImagePro Plus Software. Reported values are the mean of the percentage of MDR-1 positive signal counted in 5 fields/sample ± SE. Statistical significance vs. Placebo ##P<0.0005 ###P<0.00005; vs. DOX **P<0.005 ***P<0.0005; vs. pl-DOX §§§P<0.0005.

• 

  Figure 6. Examination of cardiotoxicity of HFn-DOX, pl-DOX and DOX a. Nanodelivery protects cardiomyocytes from DOX-induced hypertrophy. Hearts excised at day 21 (n = 3/group) from mice treated with placebo or with 1.24 mg kg−1 of DOX, pl-DOX or HFn-DOX were fixed with formalin and embedded in paraffin. Histological slides of cardiac sections stained with FITC-WGA were analyzed with ImageJ software to measure cross-section area of cardiomyocytes. Quantification was performed on at least 5 images/group, reporting the mean value of cross-section area of 250 cells/group± SE. Statistical significance vs. Placebo ###P<0.00005; vs. DOX ***P<0.00005; vs. pl-DOX §§§P<0.00005. b. Nanodelivery protects against DOX-induced mitochondrial toxicity. Hearts excised at day 21 (n = 3/group) from mice treated with placebo or with 1.24 mg kg−1 of DOX, pl-DOX or HFn-DOX were fixed with glutaraldehyde and embedded in epoxy-resin. TEM images of ultrathin heart sections of cardiac tissues acquired at 4200 magnifications were analyzed with ImageJ to measure the number of mitochondria in heart tissue. Quantification was performed on at least 9 images/group, reporting the mean mitochondria number/image ± SE.…