(T1130-02-12) Idasanutlin-Ionizable Lipid Nanocomplex (IDLIN) for Enhanced Solubility, Stability, and Anticancer Activity in p53 Sensitive Lung Cancer

Purpose: Globally, lung cancer is the most common type of cancer caused by uncontrolled cell growth in the lung tissue resulting in affecting the primary function of lungs that is providing oxygen to the body, leading to the cancer related mortality in men and women. Lung cancer is mainly categorized in two types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) accounting for 85% and 15% of the cases respectively. The overexpression of MDM2 in cancer cells inhibits the tumor suppressive effect of p53, therefore facilities the proliferation and metastasis of lung cancer cells. A novel MDM2 inhibitor – Idasanutlin showed very promising anticancer activity in vitro but it is a brick-dust molecule (Poorly soluble in both water and oil). Brick-dust molecules not only post challenges in aqueous solubility/drug delivery but also restricts development of lipid based nanoformulation. We hypothesized that hydrophobic complex between anionic idasanutlin (IDA) and ionizable lipid (DLin-DMA) will result in development of lipid nanocomplex and enhanced stability in water. Moreover, pH dependent positive charge will facilitate tumor specific delivery without toxicity in blood. In this regard, the purpose of this study is to develop, optimize and characterize the self-emulsifying nanocomplex of idasanutlin-ionizable lipid (IDLIN) and test the anticancer efficacy using various 2D and 3D cell culture assays in p53 sensitive lung cancer cells. This is the very first research in use of ionizable lipid in stabilizing anionic hydrophobic molecules and testing nanoformulation of MDMD2 inhibitor in lung cancer.

Methods: Based on saturation solubility of IDA in various organic solvents, IDLIN was prepared dissolving IDA in Dimethyl Acetamide (DMA), DLin-DMA (ionizable lipid, pKa-6.5, positive charge at lower pH and neutral at physiological pH) and preconcentrate of oil and non-ionic surfactant (1:1), where Medium Chain Triglycerides (MCT) as oil and Solutol HS 15 as surfactant was used. IDLIN, blank ionizable lipid nano-carrier (BLIN) containing DLin-DMA, DMA, MCT, Solutol HS 15 was characterized using a dynamic light scattering (DLS) particle size analyzer for particle size, polydispersity index (PDI) and zeta potential. Antiproliferative activity of IDA and IDLIN was evaluated using MTT assay in A549 and H460 cells. A549 and H460 have wild type p53 tumor suppressor protein. Multicellular 3D spheroids were treated with IDA and IDLIN every third day for up to 9 days at 1μM concentration of IDA and imaged for live/dead cells using calcein AM, ethidium homodimer and DAPI.

Results: Addition of DMA and DLinDMA resulted in significantly higher drug (IDA) loading (3%w/w) compared to conventional self-nanoemulsifying system (0.5%w/v). Moreover, formulation without ionizable lipid-DLinDMA showed very rapid precipitation of IDA from the preconcentrate indicating the dire necessity of hydrophobic ion complexing for the development of a stable system (Fig.1B). The particle size and polydispersity index of BLIN (no IDA) was 104 nm/0.214 with zeta potential of +31.5mV (in water) (Fig.2A,B). On addition of IDA i.e. IDLIN formulation zeta potential reduced to +13.1 mV which clearly indicated the reduction in free tertiary amine on the surface of IDLIN nanoglobule (Fig.2D). Complexation between anionic IDA and ionizable amine resulted in stabilization of IDA within nanoglobule. There was no change in particle size and polydispersity index was observed though. Zeta potential was nearly neutral in phosphate buffer saline and no drug precipitation was observed. In vitro efficacy of IDLIN was compared with IDA solution in DMSO.  In multicellular 3D spheroids, the treatment groups in both the cell lines A549 and H460 have shown significant difference in the diameter and area compared to the control group (Fig.3A,D). At the end of the treatment in A549, the treatment group of IDA and IDLIN showed 45.70% and 46.18% of the decrease in the area respectively, where in H460, IDA and IDLIN showed 57.64% and 59.23% of decrease in the area respectively (Fig.3C,F).  The treatment group showed a higher amount of red fluorescence compared to control group indicating large number of dead cells compared to control group (Fig.3B,E). IDA showed 0.641µM of IC50 in H460 and 3.615µM in A549 whereas IDLIN showed 0.707µM and 4.766µM of IC50 in H460 and A549, respectively (Fig.2E,F). In vitro assay showed nearly similar anticancer efficacy of IDA in DMSO and IDLIN which is expected. However, nanoformulation is more effective in vivo due to tumor selective biodistribution.

Conclusion: IDLIN – an ionizable lipid loaded nanoemulsion formulation was developed and tested for the delivery of Idasanutlin. With the emergence of novel class of anticancer molecules, a more efficient drug delivery platform is warranted for proficient delivery. Ion pairing with ionizable lipids could be potential approach in formulating nanocarrier with high drug loading, positive charge at low pH (tumor) and enhanced solubility in aqueous milleu. The potential anticancer efficacy of IDLIN in 2D and 3D in vitro NSCLC culture models has never been explored so far.


Fig. 1. (A) Mechanism of action of Idasanutlin in lung cancer cells. (B) Development and comparison of D-Lin-DMA loaded nano carrier with conventional self-nano emulsifying drug delivery system (SNEDDS). Pictorial representation of IDLIN and conventional SNEDDS system. Complexation between anionic IDA and ionizable amine resulted in stabilization of IDA within nanoglobule.


Fig. 2. (A), (B), (C) and (D) Physicochemical characterization - Dynamic light scattering graphs illustrating unimodal particle size and ζ-potential for IDLIN and BLIN respectively. In vitro cytotoxicity measured by MTT assay after 48 h treatment of IDA and IDLIN. (E) and (F) Represents the data for cell cytotoxicity treated with IDA and IDLIN in H460 and A549 respectively.


Fig. 3. Formation of multicellular tumor spheroids and their characterization. (A) Bright field microscopy images of H460 spheroids treated with IDA and IDLIN (4X, 1000 µm scale bar) (B) In H460 cells treated with IDA and IDLIN showed considerably higher apoptotic protein expression compared to control. (C) Significant reduction in area of H460 cells treated with IDA and IDLIN was observed (****p < 0.0001) (D) Bright field microscopy images of A549 spheroids treated with IDA and IDLIN (10X, 400 µm scale bar) (E) In A549 cells treated with IDA and IDLIN showed considerably higher apoptotic protein expression compared to control. (F) Significant reduction in area of A549 cells treated with IDA and IDLIN was observed (****p < 0.0001).