(T1330-03-15) Exploring Paclitaxel Loaded Adenosine Conjugated PEGylated PLGA Nanoparticles for Targeting Triple-Negative Breast Cancer

Purpose: Triple-negative breast cancer (TNBC) is a subtype of breast cancer, which is aggressive and heterogenetic in nature, thereby making it difficult to treat. As of now, Paclitaxel (PTX) is used as a first-line drug for managing TNBC [1]. But the therapeutic efficacy of PTX is low due to poor aqueous solubility, systemic toxicity and serious side effects (hypersensitivity, nephrotoxicity and hepatotoxicity) associated with solvent-based formulations (Taxol^®) [2]. Even the approved PTX nanoformulation (Abraxane^® and Genexol-PM^®) fails to provide tumor-specific delivery as they rely only on the passive targeting (EPR effect) approach. To overcome these issues, we developed PTX loaded adenosine (ADN) conjugated PEGylated Poly Lactic-co-Glycolic Acid (PLGA; 50:50) nanoparticles. Here, we wanted to explore ADN as a targeting ligand for adenosine receptors (AR) which are overexpressed on TNBC [3] (Figure 1).

Methods: Initially, PEGylated PLGA (COOH-PEG-PLGA) was synthesized by carbodiimide chemistry (EDC/NHS) using carboxy-terminated PLGA (50:50) and bifunctional PEG (NH₂-PEG-COOH). Later, COOH-PEG-PLGA was reacted with ADN using the same chemistry to form ADN-PEG-PLGA and was used to prepare nanoparticles (Figure 1). The formed intermediate and conjugate were characterized using IR and ¹H-NMR. PTX-loaded ADN-PEG-PLGA nanoparticles (PTX ADN-PEG-PLGA-NP) were prepared using optimized (stabilizer concentration, drug loading and sonication cycle) emulsion diffusion evaporation method and were characterized for their size, PDI, zeta-potential, morphology, % entrapment (%EE) and % drug-loading (%DL). The Lyophilization process for NPs with suitable cryoprotectants and their storage stability (as per ICH guidelines) were performed. At in vitro level, we evaluated hemolytic potential and % cumulative PTX release for 25 days at pH 7.4 and 5.5 respectively and performed kinetic data analysis (DDsolver) for the obtained data. At cell-culture level, IC₅₀ determination (MTT assay), cell uptake (qualitative and quantitative with inhibitors) and apoptosis index (using Annexin V-Cy3^TM Kit) were evaluated using MDA-MB-231 (human) and 4T1 (mice) TNBC cell lines [4]. At in vivo level, pharmacokinetic (using SD rats) study, anticancer efficacy (using 4T1 cell line induced tumor model in BALB/c mice), qualitative biodistribution and toxicity (liver and kidney biomarkers along with histological evaluation of organs) studies were performed [5].

Results: In IR spectra, a characteristic amide peak at 1663 cm^-1 was observed and in 1H-NMR spectra characteristic peaks for PLGA, PEG and ADN were observed further, the presence of purine peaks at 𝛿 8.13 and 8.35 ppm confirmed the successful synthesis of ADN-PEG-PLGA. For preparing PTX ADN-PEG-PLGA NPs, PVA 2% (w/v) with 7.5% PTX loading (with %EE of 78.96±2.18) formed by sonication at 35% amplitude (timer: 5-sec on-and-off, for 5 min) was found to be optimum. The optimized NPs revealed a size of 135±12 nm with PDI of 0.119±0.03 and a zeta-potential of -8.52±2.52 mV. Morphological evaluation (TEM) revealed spherical particles with smooth surfaces and uniformity. Trehalose (5%w/v) was observed to be a suitable cryoprotectant (Size_{(reconstituted)}/Size_(initial): 1.11) as it showed insignificant change in physicochemical parameters (till 6-months). The in vitro release showed 58.76±6.72% and 74.61±8.93% PTX release within 25-days at pH 7.4 and 5.5 respectively. The NPs followed Higuchi release kinetics at pH 7.4 and 5.5 but followed fickian transport at pH 7.4 and super case II transport at pH 5.5. In hemolytic study, NPs showed significantly lower hemolysis (10.27±4.72%) than free PTX (48.73±8.94%) & Intaxel^® (52.27±6.29%). NPs showed IC₅₀ values of 3.42±0.86 and 3.16±0.63 µg/mL which is ~3.7 and 3.5-fold lower than free PTX in MDA-MB-231 and 4T1 cells respectively. In cellular drug uptake study, NPs showed the highest PTX uptake, and the uptake was reduced by >50% when cells were incubated with an AR blocker (Dipyridamole). This indicated AR-mediated uptake of NPs. NPs revealed an apoptotic index of 0.93 and 1.18 which, was higher than free PTX (0.40 and 0.46) in MDA-MB-231 and 4T1cells respectively (Figure 2). The AUC_(0-α), t_1/2 and MRT values for developed NPs were significantly (P < 0.05) higher than Intaxel^® and Nanoxel^® also, t_1/2 of PTX was improved by ~5.46-fold when compared to free PTX. At the end of the anticancer study, NPs showed 0.372±0.12% tumor burden which was significantly lower than control, Intaxel^® (5.908±1.820%) and Nanoxel^® (3.168±1.050%). The biodistribution study showed tumor-specific delivery of NPs which was well corroborated by insignificant changes in the kidney (BUN & creatinine) and liver (ALT& AST) biomarkers in comparison to control and Intaxel^® but was similar to Nanoxel^® (Figure 3).

Conclusion: The results showed that the optimized PTX ADN-PEG-PLGA NPs are, hemocompatible with improved pharmaco-kinetic and -dynamic activity and exhibit reduced hepato-and nephro-toxicity compared to Intaxel^®. Also, ADN as ligand assists in improving the targetability of PTX loaded NPs. The developed system appears to be a promising strategy against TNBC.

References: [1]. Luhn, Patricia, et al. Journal of Comparative Effectiveness Research 8.14 (2019): 1173-1185.
[2]. Onetto, N., et al. Journal of the National Cancer Institute. Monographs 15 (1993): 131-139.
[3]. Swami, Rajan, et al. International Journal of Pharmaceutics 486.1-2 (2015): 287-296.
[4]. Kushwah, Varun, et al. Pharmaceutical Research 34.11 (2017): 2295-2311.
[5]. Bathara, Minal, et al. Molecular Pharmaceutics 17.7 (2020): 2473-2486.

Acknowledgements: We are thankful to Fresenius Kabi, India, for providing Paclitaxel as a gift sample for research purposes and are grateful to the Director of NIPER, S.A.S. Nagar, India, for providing necessary research facilities. We also like to thank Mr. Rahul Mahajan, NIPER S.A.S. Nagar for his technical assistance throughout the investigation. We did not receive any funding for conducting the presented research. The authors declare no conflict of interest.


Figure 1. Illustrating the synthesis scheme for ADN-PEG-PLGA and representing the outcome of the present investigation


Figure 2. Illustrating the data for mentioned in vitro and cell culture studies


Figure 3. Illustrating the data for mentioned pharmacokinetic, anticancer and toxicity studies