(M1430-03-16) Palmitoyl Carnitine-Anchored Nanoliposomes for Pancreatic Cancer and Neovasculature-Specific Delivery of Gemcitabine Elaidate

Purpose: Being the fourth most fatal malignancy worldwide, pancreatic cancer is on track to become the second leading cause of cancer-related deaths in the United States by 2030. Gemcitabine is a first-line chemotherapeutic agent for pancreatic cancer. However, due to poor aqueous solubility of gemcitabine free base (Gem Free base), its entrapment efficiency (EE) is often lower when incorporated into drug delivery carriers. Hence, Gemcitabine Elaidate (Gem Elaidate) was explored as its lipophilic derivative. Also, Gem Elaidate allows hENT1- independent intracellular delivery of gemcitabine reducing the risk of resistance development. In addition to this, we employed Palmitoyl-DL-carnitine chloride (PC) as a Protein kinase C (PKC) inhibitor which also provides a cationic surface charge to nanoliposomes for enhanced cancer cells uptake and augmented anticancer potency. The objectives of our study are: (a) to develop and characterize a PKC inhibitor-anchored Gem Elaidate-loaded PEGylated nanoliposomes (PGPL) and (b) to investigate anticancer activity of Gem Elaidate and PGPL in 2D and 3D models of pancreatic cancer.

Methods: In vitro cytotoxicity of Gem Elaidate, PC, and PGPL was assessed in PDAC cells using MTT assay. PGPL and Gem Free base-loaded liposomes (GFBL) were prepared using modified hydration method for their comparison in terms of entrapment efficiency (Fig 1A). Drug entrapment was analyzed using developed and validated HPLC method of both the drugs. Both formulations were characterized using dynamic light scattering (DLS) method to analyze particle size, polydispersity index and zeta potential. In MIA PaCa-2 cells, comparative anti-tumor efficacy of Gem Elaidate, Gem Free base and PGPL was assessed by 2D cell culture assays i.e., migration and colony formation. Gem Elaidate, Gem Free base and PGPL at 1 μM were also tested in multicellular 3D spheroid model of MIA PaCa-2 cells to mimic in vivo tumor conditions. In vitro Live/Dead Cell Assay was further performed to identify the apoptotic effect of Gem Elaidate, Gem Free base and PGPL within 3D multicellular tumor spheroids of MIA PaCa-2. Quantitative in vitro cellular uptake of both the drugs and PGPL was evaluated in MIA PaCa-2 following 4 hours of treatment. In vitro assays including vasculogenic mimicry and angiogenesis assay will be performed and hemolysis as well as microsomal assay of PGPL is currently under investigation. Western blot analysis of MIA PaCa-2 cells treated Gem elaidate and PGPL for expression of Bcl-2, p53, cleaved caspase-3, p-ERK and p-Akt will also be executed.

Results: IC₅₀ of Gem Elaidate was found to be 0.131± 0.02 µM which was significantly lower than gem free base (0.48 ± 0.04 µM) in MIA PaCa-2 cells (Fig 2A). Optimized PGPL resulted in particle size of 80 ± 2.31 nm, polydispersity index of 0.15 ± 0.05 and ζ-potential of +31.6 ± 3.54 mV (Fig 1B). Moreover, encapsulation efficiency of gem elaidate was found to be 93.25% in PGPL and that of Gem free base was significantly lower as 29.63% in GFBL (Fig 1C). A substantial change of zeta potential from − 46.4 mV in PC free nanoliposomes to +31.6 mV clearly indicated that carnitine head had occupied space on the liposomal surface. PGPL resulted in high drug loading of 5 % w/v for Gem elaidate. In vitro migration results depicted significant inhibition of scratch area on treatment with PGPL which was found to be 1.48 folds higher than that of Gem Elaidate alone (Fig 2C).Also, PGPL treated cells exhibited 3.5 folds reduction in number of colonies formed as compared to Gem Elaidate alone (Fig 2B). Furthermore, significant inhibition in growth of 3D MIA PaCa-2 spheroids treated with PGPL was observed as compared to individual drugs and control groups (Fig 3A). Most importantly, Gem Elaidate and PGPL treatment led to sustained inhibition in the growth of spheroids throughout the treatment period (Fig 3B) From the in vitro Live/Dead Cell assay, we noticed higher red fluorescence in treatment groups as compared to control confirming large number of apoptotic cells compared to control (Fig 3C).

Conclusion: In conclusion, Gem Elaidate-loaded PEGylated nanoliposomes (PGPL) were developed by modified hydration method with significantly higher drug loading, high EE, and positive surface charge for enhanced anti-pancreatic cancer activity. PGPL showed significant apoptosis and cytotoxicity in 2D cell and 3D cell culture models of pancreatic cancer cells. Hence, PGPL could be an effective and novel nano formulation for the treatment of PDAC.


Fig 1. (A) Formulation development of PGPL (B) Physicochemical characterization - Dynamic light scattering graphs illustrating unimodal particle size distribution and ζ-potential of optimized PGPL. (C) Entrapment efficiency comparison of Gem Elaidate and Gem Free base in nanoliposomes. (D) Quantitative analysis of intracellular amount of Gem Elaidate alone, Gem Free base and Gem Elaidate in PGPL in MIA PaCa-2 cells following 4 h treatment at 37℃.


Fig 2. (A) In vitro cytotoxicity of Gem Elaidate, Gem Free base, PGPL, and PC in MIA PaCa-2 cell (B) Clonogenic assay and (C) In vitro migration assay depicting microscopic images following staining with crystal violet in MIA PaCa-2 cells and percentage inhibition of migration produced by Gem Elaidate, Gem Free base and PGPL in MIA PaCa-2 cells.


Fig 3. Results for cell viability within 3D multicellular pancreatic tumor spheroids of MIA PaCa-2. (A) Representative images of spheroids treated with control, Gem Elaidate, Gem free base, and PGPL following 10 days of treatment. (B) Area of spheroids as a function of time with various treatment groups. (C) Fluorescence images depicting apoptosis of spheroids treated with various treatment groups.