Next-generation paclitaxel-nanoparticle formulation for pancreatic cancer treatment

Abstract

Pancreatic cancer (PanCa) is a major cause of cancer-related death due to limited therapeutic options. As pancreatic tumors are highly desmoplastic, they prevent appropriate uptake of therapeutic payloads. Thus, our objective is to develop a next-generation nanoparticle system for treating PanCa. We generated a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated paclitaxel loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PPNPs). This formulation exhibited optimal size (~160 nm) and negative Zeta potential (-6.02 mV), efficient lipid raft mediated internalization, pronounced inhibition in growth and metastasis in vitro, and in chemo-naïve and chemo-exposed orthotopic xenograft mouse models. Additionally, PPNPs altered nanomechanical properties of PanCa cells as suggested by the increased elastic modulus in nanoindentation analyses. Immunohistochemistry of orthotopic tumors demonstrated decreased expression of tumorigenic and metastasis associated proteins (ki67, vimentin and slug) in PPNPs treated mice. These results suggest that PPNPs represent a viable and robust platform for (PanCa).

Keywords: Paclitaxel nanoformulation.

Figure 1:. Synthesis and characterization of PLGA-PTX nanoparticles (PPNPs).

(A) Schematic overview of PPNPs synthesis. (B) DLS characterization of PLGA and PPNPs. (C) DLS characterization of zeta potential for PLGA and PPNPs. (D) FTIR spectra for free PTX, PLGA, and PPNPs. (E) XRD spectra for free PTX, PLGA, and PPNPs. (F) Topographical and physical analysis of PLGA and PPNPs by atomic force microscopy (AFM). Representative 2D and 3D images (2×2 μm²) of PLGA and PPNPs (i-ii). Modulus data for two separate samples (iii-iv).

Figure 2:. Cellular uptake of PPNPs in PanCa cells.

(A,B) Representative images of HPAF-II (Ai) and Panc-1 cells (Bi). Quantification of uptake by flow cytometry for HPAF-II (Aii) and Panc-1 (Bii). Uptake inhibitor assay for HPAF-II (Aiii) and Panc-1 (Biii) cells. Values in bar graphs represent mean±SE value of three sample in each group. *P<0.05.

Figure 3:. Effect of PPNPs on proliferation, invasion and migration of PanCa cells.

(A) Effect of PPNPs on cell viability of Panc-1, HPAF-II, and MIA PaCa-2. Values in bar graph represent mean ± SEM of 5 wells. (B) Real-time proliferation data using xCELLigence of AsPC1 cells. (C) Effect of PPNPs on clonogenic potential of PanCa cells. Representative colony images of control and PPNPs treated groups (i). Average colony counts in bar graph (ii). (D) Effect of PPNPs on invasive potential of PanCa cells. Representative images showing the effect of PPNPs on invasion of Panc-1 and AsPC1 cells through matrigel invasion assay (i). Bar graph quantification of invaded cells (ii). (E) Effect of PPNPs (24 hrs treatment) on migration of Panc-1 cells (i) Bar graph quantification of migrated cells (ii). (F) Real-time effect of PPNPs on AsPCl cells migration as shown using xCELLigence system.

Figure 4:. Effect of PPNPs on physical characteristics of PanCa cells.

Nanoindentation analyses of AsPCl (A) and PanC-1 cells. Three-dimensional images of untreated (i) and PPNPs-treated (ii) cells. Physical data collected by nanoindentation showing changes in modulus (iii) and adhesion (iv) with PPNPs. (B) Analysis of Panc-1 cells. Three-dimensional images of untreated (i) and PPNPs-treated (ii) cells. Physical data collected by nanoindentation shows changes in modulus (iii) and adhesion (iv) with PPNPs.

Figure 5:. Effect of PPNPs on cell cycle distribution and apoptosis of PanCa cells.

(A) Flow cytometric analysis of cell cycle arrest by PPNPs on Panc-1 and HPAF-II. Representative histogram images of cell cycle analysis (i). Table showing percentage of Panc-1 and HPAF-II cells in each phase of cell cycle after PPNPs treatment (ii). (B) Flow cytometric analysis of Annexin-V/7-AAD positive Panc-1 cells after treatment with PPNPs (i). Quantification of early and late apoptosis induction by PPNPs in Panc-1 cells (ii). (C) Effect of PPNPs on PARP protein cleavage in Panc-1 and HPAF-II cells as determined by Western blot analysis (i). Quantification of cleaved PARP to PARP proteins ratio calculated by the band intensity using Image J software (ii).

Figure 6:. Effect of PPNPs on pancreatic tumor growth in orthotopic xenograft mouse model.

(A) Schematic representation of schedule for dose escalation study of PPNPs in orthotopic xenograft mouse model. (B) Representative bioluminescence images of PLGA and PPNPs treated mice. (C) Quantification line graph of bioluminescence data in live mice. Values are shown as mean ± SEM with 6 mice per group. Inset images show a visual representation of tumor growth in indicated groups. (D) Representative pictures of ex vivo imaging of excised pancreatic tumor of PLGA and PPNPs treated mice. (E) Excised tumor weight of PLGA and PPNPs treated mice. Each dot represents one mouse. (F) Representative pictures of ex vivo imaging of liver, lymph nodes and lungs of PLGA and PPNPs treated mice. (G) Bar graphs indicating quantification of bioluminescence data for liver (i) and lymph nodes (ii) in PLGA and PPNPs treated mice. (H) H&E staining of pancreatic tumor of PLGA and PPNPs treated mice. Black arrows indicate pancreatic tumor. Red arrows indicate pancreatic acinar cells. Green arrows indicate necrotic area of pancreatic tumor. (I) Representative images of immunohistochemical analysis of Ki-67 in PLGA and PPNPs treated mice tissue sections.

Figure 7:. Effect of PPNPs on pancreatic cancer metastasis and drug-resistant tumors.

(A) Representative H&E staining of liver and lung sections excised from PLGA and PPNPs treated mice. Black arrows indicate metastatic foci. (B-C) Representative immunohistochemical images of vimentin and slug in excised tissues. (D) Schematic representation of treatment schedule in orthotopic xenograft tumor-bearing mice pre-exposed to PTX. (E) Representative bioluminescence images showing tumor density in PLGA and PPNPs treated mice. (F) Quantification of bioluminescence data in PLGA and PPNPs treated mice. Values are shown as mean ± SEM with 3 mice per group. (G) Excised tumor weight in PLGA and PPNPs treated mice.