(T1130-07-41) Intratumoral Delivery of Lapatinib via pH-Responsive Liposomes Embedded in a Thermosensitive In Situ Hydrogel: A Potential Chemotherapeutic Modality for Breast Cancer

Purpose: Breast cancer (BC) is not only a major health concern for women but also one of the leading causes of cancer-related deaths worldwide. Although systemic chemotherapy remains a common treatment approach, it often results in severe adverse effects due to nonspecific biodistribution, thereby limiting drug exposure to the tumor site in the breast. Among targeted therapies, Lapatinib (LPT), a tyrosine kinase inhibitor, has shown notable effectiveness against BC. However, its clinical application is significantly limited by poor water solubility, systemic toxicity, and the emergence of multidrug resistance. One of the major hurdles in treating BC with LPT is achieving sufficient drug accumulation at the tumor site without affecting healthy organs. Currently, the marketed oral formulation, TYKERB™, requires a high daily dose of 1250 mg to reach therapeutic levels, which further contributes to systemic side effects. To overcome these limitations, we propose the development of LPT-loaded, pH-responsive liposomes (LPT-Lipo) embedded within thermosensitive hydrogels. This approach aims to enhance tumor-specific drug delivery by leveraging the unique advantages of the intratumoral (i.t.) route. Specifically, the in situ-forming thermosensitive gel facilitates localized administration of LPT-Lipo, enabling sustained drug release, improved therapeutic efficacy, and reduced systemic exposure. Overall, this strategy holds promise for increasing LPT localization at the tumor site and improving patient compliance through site specific, minimally invasive treatment.

Methods: LPT-Lipo was formulated using pH-responsive phospholipids in combination with cholesterol via the thin-film hydration method. The formulation was optimized by varying the ratio of pH-sensitive phospholipids. The resulting liposomes were characterized for various physicochemical parameters. After evaluation of LPT-Lipo, optimal formulation was subjected to in vitro (cytotoxicity and cellular uptake) studies in 4T1 and MDA-MB-231 cell lines to assess its therapeutic potential. Further, the LPT-Lipo formulation was incorporated into a thermosensitive hydrogel using the cold method, employing Pluronic F127 (PF127) and Chitosan (Chit) separately as polymeric components to form LPT-Lipo@PF127 and LPT-Lipo@Chit, respectively. The hydrogels were characterized for gelation temperature, rheological properties, and in vitro drug release behavior. The concentrations of PF127 and Chitosan were adjusted to achieve the optimal gelation temperature (34-37°C) using the tube inversion method. The in vitro release of LPT-Lipo@PF127, and LPT-Lipo@Chit were conducted using the dialysis bag method (MWCO: 12-14 kDa) in acetate buffer (pH 5.5) with 0.5% tween 80 to maintain the sink conditions and compared with free LPT, and LPT-Lipo,. The optimized LPT-Lipo formulation was further assessed for in vitro studies in 4T1 and MDA-MB-231 cell lines to validate its therapeutic potential. Parallelly, in vivo anti-tumor efficacy was checked using a orthotropic model in BALB/c mice by reporting solid tumor volume regression following i.t.administration of hydrogels. Furthermore, organ histopathological evaluation, toxicity biomarker levels, and hemolysis studies were performed to check systemic toxicities. Additionally, a storage stability study was performed by periodically evaluating clarity, pH, viscosity, and drug content to ensure formulation robustness over time.

Results: The optimized LPT-Lipo formulation demonstrated a particle size distribution of (138.7 ± 1.15) nm, a polydispersity index of (0.244 ± 0.014), a zeta potential of -19.1 mV, a practical drug loading of (16.09 ± 0.11)% w/w, and an entrapment efficiency of (80.42 ± 0.78) %w/w. Morphological analysis using FE-SEM and TEM confirmed the spherical shape of the liposomes, consistent with the zeta sizer data. For hydrogel formation, LPT-Lipo@PF127 and LPT-Lipo@Chit achieved optimal gelation temperatures at polymer concentrations of 20% w/v PF127 and 2% w/v chitosan, respectively. Rheological studies revealed that the formulations maintained low viscosity and elasticity at low and room temperatures, but increased markedly at physiological temperature, facilitating in situ gelation. Importantly, the formulation maintained a viscosity below ~500 centiPoise, which supports good syringeability - a major concern for the intratumoral injection. In vitro drug release studies demonstrated a rapid release profile for free LPT (~91% within 8 hours), whereas LPT-Lipo, and hydrogels exhibited diffusion based sustained release behavior. Specifically, LPT-Lipo released ~78% of LPT (over 72 hours), while incorporation into gel matrices further extended the release to ~65% (over 72 hours) for LPT-Lipo@PF127 and ~62% (over 8 days) for Lipo@Chit. Cytotoxicity studies revealed a ~5-folds and ~4.4-folds reduction in IC₅₀ value in 4T1 and MDA-MB-231 cells, respectively for LPT-Lipo compared to free LPT. These results were further supported by quantitative cellular uptake studies, which confirmed superior internalization of LPT-Lipo in both cell lines. In vivo evaluation showed that a single intratumoral injection of LPT-Lipo@Chit exhibited much better antitumor efficacy among all treatment groups. Additionally, both the hydrogel significantly reduced major organ toxicity compared to subcutaneous administration of free LPT.

Conclusion: This study highlights the potential of LPT-Lipo embedded within thermosensitive hydrogels as an effective formulation for localized i.t. delivery in BC. The formulation demonstrated improved drug delivery, enhanced cellular uptake, and sustained drug release. In vivo studies showed significant tumor size reduction in a mouse model without notable systemic toxicity, further supporting its promise for localized chemotherapy.

References: 1. Zhang ZR, Gong T. A novel injectable phospholipid gel co-loaded with doxorubicin and bromotetrandrine for resistant breast cancer treatment by intratumoral injection. Colloids Surf B Biointerfaces. 2016 Apr 1;140:538-547.
2. Dalia S. Shaker, Mohamed A. Shaker, Anke Klingner, Mahmoud S. Hanafy, In situ thermosensitive Tamoxifen citrate loaded hydrogels: An effective tool in breast cancer loco-regional therapy, Journal of Drug Delivery Science and Technology, Volume 35, 2016, Pages 155-164, ISSN 1773-2247.
3. Zhang Y, Yang L, Ou Y, Hu R, Du G, Luo S, Wu F, Wang H, Xie Z, Zhang Y, He C, Ma C, Gong T, Zhang L, Zhang Z, Sun X. Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity. Acta Pharm Sin B. 2024 Jan;14(1):350-364.

Acknowledgements: We are grateful to the Director, NIPER SAS Nagar, India for providing facilities and infrastructure for the research work. Gratitude is also extended to Mr. Rahul Mahajan, Technical Assistant in the Department of Pharmaceutics, NIPER, S.A.S. Nagar, for his technical support in conducting scanning electron microscopy. Additionally, the authors acknowledge Mr. Vinod Kumar, Technical Supervisor Grade II in the Department of Pharmacology and Toxicology, NIPER, S.A.S. Nagar, for his technical assistance in performing transmission electron microscopy. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. The authors declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this abstract. All animal experiments were approved by the Institutional Animal Ethics Committee (IAEC/24/88), NIPER S. A. S. Nagar, and performed in accordance with CCSEA guidelines.


Figure 1: Depiction of in-situ hydrogel behavior following intratumoral injection for breast cancer treatment.


Figure 2: Illustration of optimization parameters and the final optimized formulation (highlighted with an orange rectangle), along with TEM and SEM images of LPT-Lipo and hydrogels, respectively.


Figure 3: Graphical illustration of cell viability assay plots with IC50 values, quantitative cellular uptake study, hemolytic toxicity assay, in-vitro drug release profile, and hydrogel degradation behavior.