(W1230-02-07) Docetaxel and QDs Based Nano Lipidic Theragnostic System for Cure and Real-Time Monitoring of Breast Cancer: Efficacy and Safety Studies in Rats

Purpose: Comprehensive treatment and monitoring of breast cancer in real-time using docetaxel (DTX) co-encapsulated (DCLEN; therapeutic), in solid lipid nanoparticles (SLNs), with curcumin (Cmn). Targetability and detection were achieved by decorating DCLEN with RGD peptide (DCLEN-P) and CdSe/CdS/ZnS quantum dots (QDs)

Methods: DCLEN were systematically developed (QbD) and optimized using industry amenable hot high-pressure homogenization (HPH) and readily available GRAS ingredients followed by extensive characterisation, assay, % encapsulation, in vitro release, stability studies (as per ICH guidelines),in vivo pharmacokinetics and tissue distribution in rats and its comparison with marketed product (Taxotere^®) wherever possible. The CdSe/CdS/ZnS QDs were separately synthesized and conjugated with RGD peptide employing EDC/HNS method to obtain (QDs-P). DCLEN were then decorated with QDs-P to form QDCLEN-P. System was confirmed for conjugation (UV), photoluminescence and other characteristics. Apoptotic effect of DCLEN and DCLEN-P was established in three breast cancer cell lines viz. MCF-7, MDA-MB-231 and T47 D. Uptake of FITC or QD labelled SLNs with or without peptide into cancer cells was confirmed by CLSM. Developed systems were evaluated for anticancer effects in breast tumours induced in rats using DMBA, in terms of tumour volume, histology and cancer marker CA 15-3.

Results: Optimised DCLEN were spherical, with size 317.6±30.3 nm and a significant payload of 25% DTX + 2.5% curcumin, to a total of 27.5% cumulative for both the actives. A sustained release of DTX up to 96 h was achieved as compared to Taxotere^® where DTX was released in fewer than 12h. A significant increase (p < 0.05) in AUC, AUMC, C_max and MRT was exhibited by DTX alone SLNs and DCLEN as compared to Taxotere^®. Reference to the latter, an enhanced relative bioavailability of 8.68 times and 7.13 times and 10 times mean residence time (MRT) was achieved in case of DCLEN and DTX SLN, respectively. Further a significantly lower (~6 times) deposition of DTX was observed in kidney and liver at 15 min of administration as SLNs as compared to Taxotere^®. DCLEN were stable in terms of total active content and entrapment efficiency up to 12 month storage at various conditions as per Q1A (R2) ICH guidelines. The SLNs also showed a significant (p < 0.05) photostabilising effect on Cmn upon exposure to UV light as per Q1B, ICH guidelines. An 800% reduction in IC₅₀ (MCF-7, 24 h) with DCLEN as compared to DTX SLNs; further potentiated for DCLEN-P, at 48h was observed. For MDA-MB-231 and T47 D cell line, DCLEN exhibited 200 % and 400% improved killing versus DTX SLNs, respectively. The peptide conjugated FITC-SLNs (FITC-SLN-P) and also SLNs-QDs showed significantly higher uptake in cancer cells (up to 80 times free FITC; and up to 25 times FITC-SLN), at 6h. Exciting to note was development of tunnelling tubes (TNTs) in cancer cells and accumulation of FITC-SLNs-P and SLNs-QD-P in these TNTs within 1h of treatment. DCLEN-P administered rats showed almost 21 times and 11 times reduction in breast tumours and 85% reduction in CA 15-3, in comparison to DTX SLNs and DCLEN, coupled with amelioration in tissue condition. A 170% increase in tolerability of DTX upon encapsulation in SLNs indicated safety in rats. QDCLEN-P (red spots in CLSM) were observed in breast tissue at 12h post its IV administration in rats.

Conclusion: Presented system showed significantly superior results in all the preclinical studies including 200-800% higher cytotoxicity, controlled release (96h), safety (maximum tolerated dose improved by 1.7 times; 6 times lower deposition in liver so lower chance of hepatotoxicity), efficacy (8.68 relative bioavailability; 21 times better reduction of tumour size), site specific tumour targeting and mapping post administration to rats, in comparison to popularly marketed docetaxel product (Taxotere^®). The remarkably better results are attributed to the physical and chemical composition of DCLEN. This includes: (i) protection against opsonisation and evasion of RES organs due to hydrophilic components (tween 80 and PEG 400); (ii) optimal size; > 250 nm size is optimally phagocytosed by tumour cells; (iii) co-encapsulation of Cmn alongside DTX to improve sensitivity of tumour cells to DTX and also assign its synergistic cytotoxicity; (iv) RGD peptide allowing intimate contact and better engulfment of DCLEN into cancer cells via α_vβ₃ integrin and Cmn dodging Pgp efflux of DTX. The sum total of all these factors result in a highly effective chemotherapeutic tool for cure and real time monitoring of regression of breast cancer. Presence of distinct tunneling tubes (TNTs) containing peptide conjugated systems: FITC-SLNs-P and SLNs-QD-P, within 1h of treatment establishes the ability of currently presented DCLEN to transport the anticancer agents through these channels.  TNTs are known to connect remote and maybe metastatic niches of cancers. Presence of SLNs in TNTs may thus have interesting implications. These favourable results also warrant scale-up and clinical studies in future.

References: Abu-Tayeh, H., Weidenfeld, K., Zhilin-Roth, A., Schif-Zuck, S., Thaler, S., Cotarelo, C., Tan, T. Z., Thiery, J. P., Green, J. E., Klorin, G., Sabo, E., Sleeman, J. P., Tzukerman, M., and Barkan, D. (2016). Normalizing the malignant phenotype of luminal breast cancer cells via alpha(v)beta(3)-integrin. Cell Death and Disease 7, e2491.
Adhipandito, C. F., Cheung, S. H., Lin, Y. H., and Wu, S. H. (2021). Atypical renal clearance of nanoparticles larger than the kidney filtration threshold. International Journal of Molecular Sciences 22, 11182.
Chen, S., Liang, Q., Xie, S., Liu, E., Yu, Z., Sun, L., Shin, M., C., Lee, S., J., He, H., and Yang, V., C. (2016). Curcumin based combination therapy for anti-breast cancer: from in vitro drug screening to in vivo efficacy evaluation. Frontiers of Chemical Science and Engineering 10, 383-388.
Gaigalas, A. K., DeRose, P., Wang, L., and Zhang, Y. Z. (2014). Optical properties of CdSe/ZnS nanocrystals. Journal of research of the National Institute of Standards and Technology 119, 610-628.
Herbst, R., S., and Khuri, F., R. (2003). Mode of action of docetaxel – a basis for combination with novel anticancer agents. Cancer Treatment Reviews 29, 407-415.
Kiio, T. M., and Park, S. (2021). Physical properties of nanoparticles do matter. Journal of Pharmaceutical Investigation 51, 35-51.
Liu, D., and Chen, Z. (2013). The effect of curcumin on breast cancer cells. Journal of Breast Cancer 16, 133-137.
Mansouri, K., Rasoulpoor, S., Daneshkhah, A., Abolfathi, S., Salari, N., Mohammadi, M., Rasoulpoor, S., and Shabani, S. (2020). Clinical effects of curcumin in enhancing cancer therapy: a systematic review. BMC Cancer 20, 791.
Ramalingam, G., Saravanan, K. V., Vizhi, T. K., Rajkumar, M., and Kathirvel, B. (2018). Synthesis of water-soluble and bio-taggable CdSe@ZnS quantum dots. RSC Advances 8, 8516-8527.
Vasudevan, D., Trinchi, A., Hardin, S. G., and Cole, I. S. (2015). Fluorescent heavy metal cation sensing with water dispersible 2MPA capped CdSe/ZnS quantum dots,. Journal of Luminescence 166, 88-92.

Acknowledgements: Joga Singh was supported by the ICMR senior research fellowship, New Delhi (45/78/2018-N). All animal protocols were approved by the Institutional Animal Ethics Committee

a: Triple-negative breast cancer cell line; b: Double negative breast cancer cell line; c: cancer cell line with estrogen progesterone and glucocorticoid receptors.
DCLEN: Combination SLNs co-encapsulating docetaxel and curcumin; DTX SLNs-P: peptide conjugated DTX SLNs; DCLEN-P: combination SLNs conjugated with peptide
Formulations were tested at amounts equivalent to 10nM, 20nM, 40nM, 80nM, 120nM and 160nM of DTX and at 1nM, 2nM, 4nM, and 8nM of corresponding Cmn concentrations. Combination SLNs contained curcumin:docetaxel in the ratio of 1:10.
*MIC values, $ IC50

Pictures showing reduction in tumour size in (i) animals, and in (ii) segregated tumour lump, post treatment (A) Positive control (only DMBA treatment), (B) DTX SLNs, (C) DCLEN, (D) DCLEN P and (E) % reduction in tumour volume post different treatments (positive control tumour volume was taken as 100% growth (0% reduction))
All groups are significantly different from each other (p < 0.001)

Existence of cross talk as TNTs amongst MCF-7 cells at 1 h of treatment with A) free QDs, and B) SLNs-QDs-P