LHRH-Prodigiosin Conjugates as Targeted Therapeutic Agents for Triple-Negative Breast Cancer Therapy

Introduction: Triple-negative breast cancer (TNBC) accounts for about 10-15% of all breast cancers, affecting about 13 in every 100,000 American females. It is aggressive and challenging to treat due to its lack of estrogen, progesterone, and human epidermal growth factor receptor 2 (HER2) receptors typically used to detect and treat breast cancer. Current TNBC treatments (chemotherapy, radiation therapy, surgery) lack specificity and are associated with high tumor recurrence rates. Recent efforts have been made to develop targeted therapies that interact with overexpressed receptors on TNBC cells, such as luteinizing hormone-releasing hormone (LHRH), to selectively kill cancer cells. In prior work, we have demonstrated the anticancer properties of prodigiosin, a bacterial pigment extracted from Serratia marcescens, towards TNBC cells. In this work, we present a combinatorial approach to TNBC treatment that (i) utilizes LHRH-prodigiosin conjugates as targeted chemotherapeutic agents, (ii) encapsulates LHRH-prodigiosin conjugates into poly(lactic-co-glycolic acid)/polyethylene glycol (PLGA/PEG) microparticles for sustained-release drug delivery, and (iii) incorporates LHRH-prodigiosin conjugates and gold nanoparticles into PLGA core-shell microparticles for chemo-photothermal TNBC therapy. The ultimate goals of this work are to potentiate the tumor-killing properties of prodigiosin toward TNBC cells and utilize laser-assisted plasmonic heating of gold nanoparticles to regulate the release of prodigiosin and deliver hyperthermia to suppress TNBC cell growth, kill residual cancer cells, and limit tumor recurrence.

Materials and

Methods: Prodigiosin was extracted from Serratia marcescens using our established protocols. It was then conjugated with [D-Lys6]LHRH peptide using EDC/NHS crosslinking and purified with a silica-loaded gel column chromatography system. Excess LHRH was removed using 3kDa Amicon Ultra-4 Centrifugal Filter Units. This was followed by characterization using Fourier-transform infrared spectroscopy (FTIR) and liquid chromatography/mass spectrometry (LC/MS). The LHRH-prodigiosin conjugates (LPC) were subsequently encapsulated into PLGA/PEG microparticles using a single emulsion process. Finally, the LPC drugs were incorporated into PLGA core-shell microparticles containing plasmonic gold nanoparticles. These drug delivery systems were characterized using scanning electron microscopy, transmission electron microscopy, dynamic light scattering, atomic force microscopy, and in vitro drug release studies in PBS at 37°C. The in vitro viability of a TNBC cell line (MDA-MB-231) was assessed against the drug formulations using alamar blue assay, whereas the in vivo effects of the released LPC drugs on TNBC xenograft tumors were studied in 4–6-week-old female athymic nude mice. The LPC and free drugs were administered intravenously through the tail vein of the mice induced with TNBC xenograft tumors at a dose of 10 mg/kg body weight. There were three treatment groups corresponding to 14-day, 21-day, and 28-day-old tumors and two drug injections at weekly intervals. Tumor volumes were monitored daily post-injections.

Results, Conclusions, and Discussions: The goal was to develop a combinatorial approach to targeted TNBC therapy that utilizes LHRH-prodigiosin conjugates (LPC) in various drug formulations. The synthesized LPC drugs were confirmed through FTIR and LC/MS analyses. In vitro cell viability results indicated that LPC drugs inhibited TNBC cell growth more than the free drugs after 72 h (Fig 1 a,b). The LPC drugs were also more effective in shrinking tumors in vivo, where they eliminated 14-day-old tumors and shrunk 21-day and 28-day-old tumors by approx. 90% vs. the free drugs (Fig 2). Immunofluorescence staining and RT-qPCR analysis showed that LHRH receptors were initially expressed (Fig 3 b,d) and knocked-down after siRNA transfection (Fig 3 c,e,f). Afterward, there were no differences in drug-induced cytotoxicity between TNBC cells with or without knock-down receptors (Fig 1c), indicating that the LPC drug-LHRH receptor attachment allowed for targeted drug delivery to the cancer cells. This was also confirmed by AFM adhesion force measurements between drugs and xenograft tumors, where the LPC drugs exhibited greater than 2-fold adhesion forces than the free drugs.

After showing that LPC drugs selectively target TNBC, we incorporated them into PLGA/PEG microparticles to prolong their release. Drug release studies indicated that ~60% of the drugs were released within 40 days (Fig 4), and the released LPC drugs inhibited TNBC cell growth more than the free drugs. The microparticles were then deployed in vivo to kill residual cancer cells, following the resection of a 4-week-old xenograft tumor, to suppress locoregional tumor recurrence in athymic nude mice. The microparticles were placed in the resected tumor region, and after 8 weeks, there was no tumor recurrence, whereas the control mice had tumor recurrence and metastasis. Finally, LPC and gold nanoparticle-loaded core-shell microparticles (Fig 5 a,b,c) released ~40% of drugs within 30 days, significantly inhibiting cell growth. Treatment with a laser beam (808 nm) generated photothermal heating that increased drug release by ~ 1.5 times (Fig 5d), which in turn increased TNBC inhibition. Ultimately, we anticipate that our combinatorial approach will create a new standard of care for TNBC therapy that combines targeted drug delivery and photothermal therapy.

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