Self-Associating Poly(ethylene oxide)-<i>block</i>-poly(α-carboxyl-ε-caprolactone) Drug Conjugates for the Delivery of STAT3 Inhibitor JSI-124: Potential Application in Cancer Immunotherapy

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) in tumor cells and tumor associated dendritic cells (DCs) plays a major role in the progression of cancer. JSI-124 (cucurbitacin I) is a potent inhibitor of STAT3; however, its poor solubility and nonspecificity limit its effectiveness in cancer immunotherapy. In order to achieve a nanocarrier for solubilization and passive targeting of JSI-124 to tumor cells and tumor associated DCs, the drug was chemically conjugated to pendent COOH groups of self-associating poly­(ethylene oxide)-<i>block</i>-poly­(α-carboxylate-ε-caprolactone) (PEO-<i>b</i>-PCCL). Developed PEO-<i>b</i>-P­(CL-JSI-124) conjugates self-assembled to polymeric micelles of 40 nm size range with negligible drug release under physiological mimicking conditions. The conjugation of JSI-124 to PEO-<i>b</i>-PCCL was confirmed by ¹H NMR, thin layer chromatography (TLC), and HPLC with a conjugation of 8.9% w/w of the polymer. As expected, JSI-124 nanoconjugates showed lower potency in p-STAT3 inhibition and direct anticancer activity in B16-F10 melanoma cells. Interestingly, JSI-124 nanoconjugates were more powerful than free drug in reducing the level of p-STAT3 in tumor exposed bone marrow derived dendritic cells (BMDCs). The JSI-124 nanoconjugates were also significantly more active than free drug in reversing the immunosuppressive effect of B16-F10 tumor and led to significantly better phenotypical and functional stimulation of tumor exposed immature BMDCs in the presence of immune adjuvants like LPS and CpG. Our findings points to great promise for PEO-<i>b</i>-P­(CL-JSI-124) micelles for modulation of immunosuppressive microenvironment in melanoma tumors, implicating application of this strategy in cancer immunotherapy

Proteolytically Stable Cancer Targeting Peptides with High Affinity for Breast Cancer Cells

Cancer cell targeting peptides have emerged as a highly efficient approach for selective delivery of chemotherapeutics and diagnostics to different cancer cells. However, the use of α-peptides in pharmaceutical applications is hindered by their enzymatic degradation and low bioavailability. Starting with a 10-mer α-peptide <b>18</b> that we developed previously, here we report three novel analogues of <b>18</b> that are proteolytically stable and display better (up to 3.5-fold) affinity profiles for breast cancer cells compared to <b>18</b>. The design strategy involved replacement of two or three amino acids in the sequence of <b>18</b> with d-residues or β³-amino acids. Such replacement maintained the specificity for cancer cells (MDA-MB-435, MDA-MB-231, and MCF-7) with low affinity for control noncancerous cells (MCF-10A and HUVEC), showed an increase in secondary structure, and rendered the analogues completely stable to human serum and liver homogenate from mice. The three analogues are potentially safe with minimal cellular toxicity and are efficient targeting moieties for specific drug delivery to breast cancer cells. The strategy used here may be adapted to develop peptide analogues that will target other cancer cell types

Breast Cancer Targeting Peptide Binds Keratin 1: A New Molecular Marker for Targeted Drug Delivery to Breast Cancer

The biomarkers or receptors expressed on cancer cells and the targeting ligands with high binding affinity for biomarkers play a key role in early detection and treatment of breast cancer. The breast cancer targeting peptide p160 (12-mer) and its enzymatically stable analogue <b>18-4</b> (10-mer) showed marked potential for breast cancer drug delivery using cell studies and animal models. Herein, we used affinity purification, liquid chromatography–tandem mass spectrometry, and proteomics to identify keratin 1 (KRT1) as the target receptor highly expressed on breast cancer cells for p160 peptide(s). Western blot and immunocytochemistry in MCF-7 breast cancer cells confirmed the identity of KRT1. We demonstrate that the p160 or <b>18-4</b> binding to MCF-7 breast cancer cells is dependent on the expression of KRT1, and we confirm peptide-KRT1 binding specificity using SPR experiments (<i>K</i>_d ∼ 1.1 μM and 0.98 μM for p160 and <b>18-4</b>, respectively). Furthermore, we assessed the ability of peptide <b>18-4</b> to improve the cellular uptake and anticancer activity of a pro-apoptotic antimicrobial peptide, microcin J25 (MccJ25), in breast cancer cells. A covalent conjugate of peptide <b>18-4</b> with MccJ25 showed preferential cytotoxicity toward breast cancer cells with minimal cytotoxicity against normal HUVEC cells. The conjugate inhibited the growth of MDA-MB-435 MDR multidrug-resistant cells with an IC₅₀ comparable to that of nonresistant cells. Conjugation improved selective cellular uptake of MccJ25, and the conjugate triggered cancer cell death by apoptosis. Our findings establish KRT1 as a new marker for breast cancer targeting. Additionally, it pinpoints the potential use of antimicrobial lasso peptides as a novel class of anticancer therapeutics

Proteolytically Stable Cyclic Decapeptide for Breast Cancer Cell Targeting

Starting with a previously reported linear breast cancer targeting decapeptide WxEAAYQkFL, here we report the synthesis of a novel cyclic peptide analogue cyclic WXEAAYQkFL. The N- to C-terminus amide cyclized peptide with one d-amino acid (k) displayed higher uptake by breast cancer cells, with minimal uptake by the noncancerous cells compared to the linear peptide with two d-amino acids (x and k), and was stable toward proteolytic degradation. When immobilized on gold microcantilever surface, the cyclic peptide was able to capture breast cancer cells specifically and sense samples with ≥25 cancer cells/mL. Animal studies using mice carrying orthotopic breast MDA-MB-231 tumors showed that the cyclic peptide preferentially accumulates in tumor (2 h after injection) and is rapidly cleared from all other organs except kidneys and liver. The study highlights the discovery of a novel proteolytically stable cyclic peptide that can be used for targeted drug delivery or for enumerating circulating breast tumor cells

Assessment of competitive PS- or apoptosis-binding of apoptosis-targeting PEG-<i>b</i>-PCL micelles carrying DiR by PS saturation with free peptides <i>in vitro</i>.

<p>Results are presented as %FLI DiR molecules bound on plates and %FLI from DiR molecules bound on ES-2-luc ovarian tumor spheroids. BLI from ES-2-luc cells and FLI from DiR molecules were quantified using Xenogen IVIS 200 Series. (A) Competitive binding test of apoptosis-targeting PEG-<i>b</i>-PCL micelles carrying DiR (1.0 µM peptide and 500 nM DiR) to PC- or PS-coated 96-well plates (total 200 µM of phospholipid). (B) Competitive binding test of apoptosis-targeting PEG-<i>b</i>-PCL micelles carrying DiR to apoptosis-induced ES-2-luc ovarian tumor spheroids (** <0.01, *** <0.001).</p

Evaluation of apoptosis-targeted interval debulking surgery in ES-2-luc-bearing xenograft model.

<p>Evaluation of apoptosis-targeted interval debulking surgery in ES-2-luc-bearing xenograft model.</p

Illustration of Peritoneal Cancer Index (PCI) by Sugarbaker.

<p>(A) A composite score (0–3) of lesion size in abdomino-pelvic regions (0–12). (B) The scoring system of PCI adapted from Sugarbaker.</p

Polymeric Micelles for Apoptosis-Targeted Optical Imaging of Cancer and Intraoperative Surgical Guidance

<div><p>In a two-step strategy, an intraperitoneal (IP) injection of poly(ethylene glycol)-<i>block</i>-poly(ε-caprolactone) (PEG-<i>b</i>-PCL) micelles containing paclitaxel (PTX), cyclopamine (CYP), and gossypol (GSP) at 30, 30, and 30 mg/kg, respectively, debulked tumor tissues by 1.3-fold, based on loss of bioluminescence with <10% body weight change, and induced apoptosis in peritoneal tumors when used as neoadjuvant chemotherapy (NACT) in an ES-2-luc-bearing xenograft model for ovarian cancer. In a second step, a single intravenous (IV) injection of apoptosis-targeting GFNFRLKAGAKIRFGS-PEG-<i>b</i>-PCL micelles containing a near-infrared (NIR) fluorescence probe, DiR (1,1′-dioctadecyltetramethyl indotricarbocyanine iodide), resulted in increased peritoneal DiR accumulation in apoptosis-induced ES-2-luc tumor tissues (<i>ex vivo</i>) by 1.5-fold compared with DiR molecules delivered by methoxy PEG-<i>b</i>-PCL micelles (non-targeted) at 48 h after IV injection in a second step. As a result, a tandem of PEG-<i>b</i>-PCL micelles enabled high-resolution detection of <i>ca</i>. 1 mm diameter tumors, resulting in resection of approximately 90% of tumors, and a low peritoneal cancer index (PCI) of <i>ca</i>. 7. Thus, a tandem of PEG-<i>b</i>-PCL micelles used for NCAT and NIR fluorescence imaging of therapy-induced apoptosis for intraoperative surgical guidance may be a promising treatment strategy for metastatic ovarian cancer.</p></div

Schematic illustration of two-step strategy for neoadjuvant therapy, apoptosis-targeted optical imaging and intraoperative surgical guidance, enabled by a tandem of PEG-<i>b</i>-PCL micelles.

<p>Schematic illustration of two-step strategy for neoadjuvant therapy, apoptosis-targeted optical imaging and intraoperative surgical guidance, enabled by a tandem of PEG-<i>b</i>-PCL micelles.</p

Fluorescence images of carcass of ES-2-luc-bearing mice at different surgical stages and locations obtained by Fluobeam.

<p>Fluorescence images of carcass of ES-2-luc-bearing mice at different surgical stages and locations obtained by Fluobeam.</p