Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer

Abstract

Due to its late diagnosis and dismal prognosis, pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating solid malignancies, with only 9% of patients surviving after being diagnosed. A multidrug chemotherapeutic regimen FOL-F-IRIN-OX (combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) offers survival benefits superior to that of gemcitabine single agent, but the treatment-related side effects are also severe. To overcome this therapeutic barrier, we developed polymeric micelles bearing active formats of irinotecan and oxaliplatin, SN38 and 1,2-diaminocyclohexane‑platinum (II), DACHPt. Crosslinked micelles were prepared using amphiphilic PEG-b-poly(L-glutamic acid)/SN38 conjugates and subsequently loaded with DACHPt. The dual drug-loaded micelles exhibited improved colloidal stability, prolonged drug release and remarkable cytotoxicity in human pancreatic cancer cell lines and Kras^G12D; Trp52^R172H/+; Pdx-1 Cre murine tumor organoids models. In vivo, (SN38 + DACHPt)-loaded micelles displayed superior antitumor and antimetastatic activities without impairing safety. Our results suggest that nanomedicine mimicking irinotecan and oxaliplatin as parts of FOLFIRINOX regimen may further improve the feasibility of this multidrug treatment for patients with advanced pancreatic cancer.

Keywords: Combination therapy; DACHPt; FOLFIRINOX; Irinotecan; Oxaliplatin; Pancreatic cancer; Polymeric micelles; SN38.

Figure 1.

Schematic illustration of dual drug-loaded micelle preparation.

Figure 2.

Drug release profiles of (A) SN38 and (B) Pt from (SN38 + DACHPt)/m (1 :1 w/w) in PBS (pH 7.4), ABS (pH 5.5) and PBS in the presence of 10% FBS at 37 °C. Data are expressed as the mean ± SD (n = 3). Insert in (A) shows a comparison of SN38 release profiles from SN38/m and (SN38 + DACHPt)/m in PBS. (C) Average percentage of unbound fraction (fu) of each drug in human whole blood post incubation with (SN38 + DACHPt)/m or combination of free drugs (IRIN + OX) for 2 and 6 hours, at 37 °C. Data are presented as the mean (fu) ± SD (n = 3) and compared by one-way ANOVA with Bonferroni’s multiple comparison test. (**p < 0.01, ****p < 0.0001, ns - not significant).

Figure 3.

Cellular uptake of Cy5-labeled SN38/m and (SN38 + DACHPt)/m in CD18/HPAF cells as a function of incubation time at 37 °C as investigated by flow cytometry and expressed in (A) % gated cells and (B) mean fluorescence intensity (MFI). (C) Representative confocal images of the uptake at 60 min post-treatment with micelles and magnified images of the boxed areas (red). White, Cy5; blue, DAPI. Scale bars represent 20 µm. (D) Effect of endocytosis inhibitors on the uptake of drug-loaded micelles. Cells were pretreated with corresponding inhibitors or at 4°C for 30 minutes followed by adding Cy5-labeled SN38/m or (SN38 + DACHPt)/m at concentration of 40 µM based on SN38 equivalents and incubated for another 60 min. The relative cell uptake (%) represents the uptake of micelles by cells treated with endocytic inhibitors normalized by the uptake by nontreated ones. Data presented as mean ± SD (n = 3). Statistical comparisons between the control group (no inhibitor) and the corresponding inhibitor group were determined by Student t-test (*p < 0.05, **p < 0.01, ^#p < 0.001, ^##p < 0.0001).

Figure 4.

Drug-loaded micelle formulations suppress colony-formation ability and migration of PC cells. (A, B) Colony formation assay was performed in PANC-1 and CD18/HPAF cells that were treated with free drugs and drug-loaded micelle formulations for 48 h at indicated concentrations based on OX equivalents. (C) Inhibition of the migration of PC cells as determined by wound healing assay. Cells were treated with various drug formats for 24 h at concentration of 60 nM based on OX equivalents. Data presented as mean ± SD (n = 3). Statistical significance was determined using one-way ANOVA with Bonferroni’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001, ns - not significant).

Figure 5.

(SN38 + DACHPt)/m treatment inhibit growth of mouse pancreatic tumor organoids. (A) Representative light microscope images of tumor organoids treated with IRIN + OX (2:1) combination, SN38/m, or (SN38 + DACHPt)/m on Day 0 and Day 3. (B) The effect of various treatments on the growth of tumor organoids on Day 3. The tumor organoids were treated with free drugs or drug-loaded micelles for 3 days at concentration of 10 nM based on OX equivalents. Data are expressed as mean ± SD (n = 5), *p < 0.05, **p < 0.01.

Figure 6.

(SN38 + DACHPt)/m inhibit growth of orthotopic CD18/HPAF human pancreatic cancer xenografts. (A) Tumor inoculation and treatment schedule. (B) Changes in tumor burden as assessed by bioluminescence imaging (expressed in relative radiance units, R_t/R₀, to the day when treatment was initiated for individual animal) following treatments with 5% dextrose (control), IRIN + OX combination (IRIN 8 mg/kg, OX 4 mg/kg), SN38/m (SN38 4 mg/kg), SN38/m plus free OX(SN38 4 mg/kg, OX 4 mg/kg), and (SN38 + DACHPt)/m (SN38 4 mg/kg, OX equiv. 4 mg/kg). Insert represents tumor weights at the experimental endpoint (day 36). Data are presented as mean ± SEM (n= 8). (C) Body weight changes throughout the experiment. (D) Blood and tissue distribution of Pt in different treatment groups as determined by ICP- MS. Mice were sacrificed at day 14 (n = 3). (E) The number of metastatic sites at distant organs in the various treatment groups. Each point corresponds to an individual animal. Quantification of (F) Ki67 positive and (G) caspase-3 positive cells in tumor tissue from mice from various groups (n = 3, based on the analysis of the whole tumor slice). Statistical significance was determined using one-way ANOVA with Turkey’s multiple comparison test (*p < 0.05, **p < 0.01, ***p < 0.001).

Figure 7.

Morphological changes in CD18/HPAF after different treatments. (A) Sirus Red staining of the collagen and quantification of staining; (B) IHC staining of α-SMA (green) and blood vessel (CD31, red) with nuclear counterstain (blue), expression levels were quintified using ImageJ software (random fields for each tumor section, n = 8); (C) density of the intact and closed blood vessels with lumen > 5 mm were quantified using Definiens Tissue Studio software. *p < 0.05, **p<0.01, ***p<0.001.