(W1030-01-02) Harnessing the Potential of Self-Assembled Hyaluronic Acid Nanoparticles for Tumor Targeted Co-Delivery of Betulinic Acid and Paclitaxel

Purpose: Triple negative breast cancer is the most aggressive phenotype, with a heightened propensity for metastasis, relapse, and poor survival rates. Due to the lack of targeted therapy, conventional chemotherapeutics, such as paclitaxel (PTX), are widely used clinically, acting via inducing cell cycle arrest and apoptosis. However, challenges such as tumor heterogeneity and the emergence of drug resistance render monotherapy ineffective. Betulinic acid (BtA), a pentacyclic triterpenoid, possesses anti-tumor properties, induces cell death via the mitochondrial apoptotic pathway, and has been shown to sensitize tumor cells to chemotherapeutics [1–3]. Hence, this study aims to explore the potential of hyaluronic acid (HA) prodrug-based self-assembled nanoparticles (HA-BtA-PTX NPs) for tumor targeted co-delivery of BtA and PTX (Figure 1). BtA, being hydrophobic, is covalently linked to the hydrophilic HA via an amide bond, forming an amphiphilic conjugate that self-assembles into nanoparticles in an aqueous milieu, encapsulating PTX in its hydrophobic core. The proposed delivery system provide active tumor targeting due to the high binding affinity of HA to the CD44 receptor [4]. Moreover, enzymes like hyaluronidase (Hyal-1) present in the tumor endosomes degrade the carrier structure [5,6], facilitating the drug release at the tumor site, thus improving the therapeutic index while minimizing toxicity to normal cells.

Methods: The HA-BtA conjugate was synthesized using carbodiimide chemistry, linking HA's carboxyl group to BtA's amino group, and characterized by ¹H NMR and FT-IR. Subsequently, the conjugate's degree of substitution (DS) and critical aggregation concentration (CAC) were assessed via UV/VIS spectroscopy and pyrene assay. Hydrolytic stability was determined in PBS pH 7.4 and MDA-MB-231 cell lysate. The HA-BtA-PTX NPs were prepared through a self-assembly approach and characterized for critical quality attributes. Further the nanoparticles were evaluated for drug release at pH 7.4 and in the presence of Hyal-1 enzyme at pH 5.5. Cellular uptake mechanism was studied in MDA-MB-231 cells, with or without a competitive inhibitor (HA 10 mg/mL). In vitro assays including cytotoxicity, apoptosis, and JC-1 were performed in MDA-MB-231 and 4T1 cells to assess therapeutic efficacy.  In vivo, the pharmacokinetic study was performed in SD rats to evaluate the circulation dynamics of HA-BtA-PTX NPs. The anti-tumor efficacy of developed HA-BtA-PTX NPs was assessed in 4T1-bearing female Balb/C mice. Furthermore, the HA-BtA-PTX NPs were evaluated for hemocompatibility and organ toxicity via biomarker analysis and histological analysis.

Results: The formation of HA-BtA conjugate was confirmed by ¹H NMR and FT-IR. In the ¹H NMR spectrum, characteristic peaks were observed at 1.0-2.0 ppm corresponding to the CH₃-CH₂ group of BtA, and at 6.8-8 ppm corresponding to the -CONH group in the HA-BtA conjugate. Additionally, in the FT-IR spectra, peaks at 1637 cm^-1 and 1554 cm^-1, corresponding to the amide I and amide II bands respectively, further confirmed successful synthesis of the conjugate. The HA-BtA conjugate exhibited 9.86 mol% DS of BtA and low CAC value of 44 µg/mL. Moreover, it showed 14% hydrolysis in PBS, indicating physiological stability, and 47% hydrolysis in MDA-MB-231 cell lysate, signifying free BtA release in tumor cells. HA-BtA-PTX NPs were spherical, with a particle size of 181.06±5.74 nm and a PDI of 0.24±0.013. These NPs had 8.28% drug loading and 82.67% encapsulation efficiency of PTX. In vitro release studies showed a sustained release profile, with 69% cumulative drug release at 72 h at pH 7.4. In the presence of Hyal-I, accelerated drug release was observed, with cumulative drug release reaching 82% in 72 h. The HA-BtA-NPs were least hemotoxic (5.83%) than free PTX+BtA (26.58%). Cellular uptake studies showed a greater internalization of HA-BtA-PTX NPs compared to the free drug, facilitated via CD44 receptor-mediated endocytosis. In vitro, the HA-BtA NPs showed higher cytotoxicity, apoptotic induction, and reduction in mitochondrial membrane potential compared to PTX alone, attributed to the synergistic effect of PTX and BtA, along with enhanced cellular uptake (Figure 2). Pharmacokinetic studies revealed an improved profile for HA-BtA-PTX NPs, with 2.28-, 3.02-, and 2.62-fold higher AUC, half-life, and MRT respectively compared to free PTX. Notably, in a 4T1 tumor model, HA-BtA-PTX NPs exhibited superior antitumor efficacy, with an 86% tumor inhibition rate. Further, the toxicity study revealed the insignificant change in hepatotoxic markers (ALT and AST) and nephrotoxic markers (BUN, and creatinine). Histological analysis further confirmed their safety profile, showing no signs of toxicity in the liver, kidney, and spleen (Figure 3).

Conclusion: The nanoparticles demonstrated high drug loading, enzyme-responsive release, CD44 targeting ability, improved pharmacokinetics, enhanced anti-tumor efficacy, and biocompatibility. These results highlight the promising potential of HA-BtA-PTX NPs for tumor targeted co-delivery of chemotherapeutics with a non-toxic profile. This research paves the way for future investigations into polysaccharides- based active drug nanocarriers in combination cancer therapy.

References: [1] Y. Cai, Y. Zheng, J. Gu, S. Wang, N. Wang, B. Yang, F. Zhang, D. Wang, W. Fu, Z. Wang, Betulinic acid chemosensitizes breast cancer by triggering ER stress-mediated apoptosis by directly targeting GRP78, Cell Death Dis 9 (2018) 1–16.
[2] S. Fulda, Betulinic Acid for Cancer Treatment and Prevention, Int J Mol Sci 9 (2008) 1096–1107.
[3] S. Fulda, K.-M. Debatin, Sensitization for Anticancer Drug-Induced Apoptosis by Betulinic Acid, Neoplasia 7 (2005) 162–170.
[4] W. Li, X. Yi, X. Liu, Z. Zhang, Y. Fu, T. Gong, Hyaluronic acid ion-pairing nanoparticles for targeted tumor therapy, Journal of Controlled Release 225 (2016) 170–182.
[5] J.-Y. Lee, U. Termsarasab, M.Y. Lee, D.-H. Kim, S.Y. Lee, J.S. Kim, H.-J. Cho, D.-D. Kim, Chemosensitizing indomethacin-conjugated chitosan oligosaccharide nanoparticles for tumor-targeted drug delivery, Acta Biomater 57 (2017) 262–273.
[6] S.W. Kim, K.T. Oh, Y.S. Youn, E.S. Lee, Hyaluronated nanoparticles with pH- and enzyme-responsive drug release properties, Colloids and Surfaces B: Biointerfaces 116 (2014) 359–364.

Acknowledgements: The authors are thankful to the Director NIPER, S.A.S. Nagar, India for providing necessary research facilities. The authors would also like to thank Mr. Rahul Mahajan, NIPER S.A.S. Nagar for his technical assistance throughout the research. The authors did not receive any funding for conducting the present research. The authors declare no conflict of interest.


Figure 1. Pictorial presentation of the purpose and outcomes of the present investigation.


Figure 2. Illustration of data mentioned for HA-BtA-NPs characterization and cell culture studies.


Figure 3. Illustration of data mentioned for in vivo pharmacokinetic study, anti-tumor efficacy study and toxicity study.