Federico Perche

Enhanced anticancer activity of nanopreparation containing an MMP2-sensitive PEG-drug conjugate and cell-penetrating moiety

Significance The clinical outcomes of anticancer drugs are compromised by their poor physicochemical properties, low tumor targeting, insufficient bioavailability, and side effects. Matrix metalloproteinase 2 (MMP2) has been found overexpressed in most cancers and responsible for tumor cell proliferation and metastasis. In this study, a self-assembling MMP2-sensitive paclitaxel-containing micellar nanopreparation was developed. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in a collaborative fashion to design this nanoparticle. The nanopreparation showed superior tumor targeting, cell internalization, and antitumor efficacy over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells. In response to the challenges of cancer chemotherapeutics, including poor physicochemical properties, low tumor targeting, insufficient tumor cell internalization/bioavailability, and side effects, we developed a unique tumor-targeted micellar drug-delivery platform. Using paclitaxel as a model therapeutic, a nanopreparation composed of a matrix metalloproteinase 2 (MMP2)-sensitive self-assembly PEG 2000-paclitaxel conjugate (as a prodrug and MMP 2-sensitive moiety), transactivating transcriptional activator peptide-PEG1000-phosphoethanolamine (PE) (a cell-penetrating enhancer), and PEG1000-PE (a nanocarrier building block) was prepared. Several major drug delivery strategies, including self-assembly, PEGylation, the enhanced permeability and retention effect, stimulus sensitivity, a cell-penetrating moiety, and the concept of prodrug, were used in design of this nanoparticle in a collaborative manner. The nanopreparation allowed superior cell internalization, cytotoxicity, tumor targeting, and antitumor efficacy in vitro and in vivo over its nonsensitive counterpart, free paclitaxel and conventional micelles. This uniquely engineered nanoparticle has potential for effective intracellular delivery of drug into cancer cells.

Recent Trends in Multifunctional Liposomal Nanocarriers for Enhanced Tumor Targeting

Liposomes are delivery systems that have been used to formulate a vast variety of therapeutic and imaging agents for the past several decades. They have significant advantages over their free forms in terms of pharmacokinetics, sensitivity for cancer diagnosis and therapeutic efficacy. The multifactorial nature of cancer and the complex physiology of the tumor microenvironment require the development of multifunctional nanocarriers. Multifunctional liposomal nanocarriers should combine long blood circulation to improve pharmacokinetics of the loaded agent and selective distribution to the tumor lesion relative to healthy tissues, remote-controlled or tumor stimuli-sensitive extravasation from blood at the tumors vicinity, internalization motifs to move from tumor bounds and/or tumor intercellular space to the cytoplasm of cancer cells for effective tumor cell killing. This review will focus on current strategies used for cancer detection and therapy using liposomes with special attention to combination therapies.

Accumulation and toxicity of antibody-targeted doxorubicin-loaded PEG-PE micelles in ovarian cancer cell spheroid model.

We describe the evaluation of doxorubicin-loaded PEG-PE micelles targeting using an ovarian cancer cell spheroid model. Most ovarian cancer patients present at an advanced clinical stage and develop resistance to standard of care platinum/taxane therapy. Doxorubicin is also approved for ovarian cancer but had limited benefits in refractory patients. In this study, we used drug-resistant spheroid cultures of ovarian carcinoma to evaluate the uptake and cytotoxicity of an antibody-targeted doxorubicin formulation. Doxorubicin was encapsulated in polyethylene glycol-phosphatidyl ethanolamine (PEG-PE) conjugated micelles. The doxorubicin-loaded PEG-PE micelles (MDOX) were further decorated with a cancer cell-specific monoclonal 2C5 antibody to obtain doxorubicin-loaded immunomicelles (2C5-MDOX). Targeting and resulting toxicity of doxorubicin-loaded PEG-PE micelles were evaluated in three dimensional cancer cell spheroids. Superior accumulation of 2C5-MDOX compared to free doxorubicin or untargeted MDOX in spheroids was evidenced both by flow cytometry, fluorescence and confocal microscopy. Interestingly, even higher toxicity was measured by lactate dehydrogenase release and terminal deoxynucleotidyl transferase dUTP nick end labeling of targeted doxorubicin micelles in Bcl-2 overexpressing adriamycin-resistant spheroids. Overall, these results support use of spheroids to evaluate tumor targeted drug delivery.

Polymeric micelles containing reversibly phospholipid-modified anti-survivin siRNA: a promising strategy to overcome drug resistance in cancer

The discovery that survivin, a small anti-apoptotic protein, is involved in chemoresistance, opens a new scenario to overcome the drug resistance in cancer. It was shown that siRNA can efficiently inhibit the expression of survivin in cancer cells. However, the clinical use of siRNA is still hampered by an unfavorable pharmacokinetic profile. To address this problem, earlier we developed a novel system to deliver siRNA into cancer cells. Namely, we reversibly modified the survivin siRNA with a phosphothioethanol (PE) portion via a reducible disulfide bond and incorporated the resulting siRNA-S-S-PE conjugate into nanosized polyethyelene glycol 2000-phosphatidyl ethanolamine (PEG2000-PE)-based polymeric micelles (PM), obtaining survivin siRNA PM. The activity of these nanopreparations was evaluated by survivin protein down-regulation, tumor cell growth inhibition, and chemosensitization of the treated tumor cells to paclitaxel (PXL). We found a significant decrease of cell viability and down-regulation of survivin protein levels after treatment with survivin siRNA PM in several cancer cell lines. In addition, the down-regulation of survivin by treating cells with survivin siRNA PM, elicited a significant sensitization of the cells to PXL, in both sensitive and resistant cancer cell lines. Finally, we demonstrated successful co-delivery of PXL and survivin siRNA in the same PM leading to superior therapeutic activity compared to their sequential administration. Our results support the use of this new platform for the treatment of the most aggressive tumors.

Octa-arginine-modified pegylated liposomal doxorubicin: An effective treatment strategy for non-small cell lung cancer

The present study aims to evaluate the efficacy of octa-arginine (R8)-modified pegylated liposomal doxorubicin (R8-PLD) for the treatment of non-small cell lung cancer, for which the primary treatment modality currently consists of surgery and radiotherapy. Cell-penetrating peptide R8 modification of Doxorubicin-(Dox)-loaded liposomes was performed by post-insertion of an R8-conjugated amphiphilic PEG-PE copolymer (R8-PEG-DOPE) into the liposomal lipid bilayer. In vitro analysis with the non-small cell lung cancer cell line, A549 confirmed the efficient cellular accumulation of Dox, delivered by R8-PLD compared to PLD. It led to the early initiation of apoptosis and a 9-fold higher level of the apoptotic regulator, caspase 3/7 (9.24±0.34) compared to PLD (1.07±0.19) at Dox concentration of 100 μg/mL. The treatment of A549 monolayers with R8-PLD increased the level of cell death marker lactate dehydrogenase (LDH) secretion (1.2±0.1 for PLD and 2.3±0.1 for R8-PLD at Dox concentration of 100 μg/mL) confirming higher cytotoxicity of R8-PLD than PLD, which was ineffective under the same treatment regimen (cell viability 90±6% in PLD vs. 45±2% in R8-PLD after 24h). R8-PLD had significantly higher penetration into the hypoxic A549 tumor spheroids compared to PLD. R8-PLD induced greater level of apoptosis to A549 tumor xenograft and dramatic inhibition of tumor volume and tumor weight reduction. The R8-PLD treated tumor lysate had a elevated caspase 3/7 expression than with R8-PLD treatment. This suggested system improved the delivery efficiency of Dox in selected model of cancer which supports the potential usefulness of R8-PLD in cancer treatment, lung cancer in particular.

Cancer cell spheroids as a model to evaluate chemotherapy protocols

To determine whether the spheroid culture can be used to evaluate drug efficacy, we have evaluated the toxicity of free or carrier-associated doxorubicin as a single drug or in combination with other antineoplastic agents using the spheroid cultures of drug-resistant cancer cells. Paclitaxel, cisplatin, dexamethasone, mitoxantrone, sclareol or methotrexate were used in combination with doxorubicin. The effect of the treatment protocols on free, micellar and liposomal doxorubicin accumulation in spheroids and on resulting toxicity was evaluated by fluorescence and lactate dehydrogenase release, respectively. Enhanced doxorubicin accumulation and toxicity were observed after spheroid pretreatment with mitoxantrone or paclitaxel. Effects of the drug combination with doxorubicin were sequence dependent, use of doxorubicin as the first drug being the least inducer of toxicity. Finally, spheroids were recognized by a cancer cell-specific antibody. Our results suggest the usefulness of spheroids to evaluate chemotherapy combinations.

Hypoxia-Responsive Copolymer for siRNA Delivery.

A wide variety of nanomedicine has been designed for cancer therapy. Herein, we describe the synthesis and evaluation of a hypoxia-responsive copolymer for siRNA delivery (Perche et al., Angew Chem Int Ed Engl 53:3362-3366, 2014). The synthesis is achieved using established coupling chemistry and accessible purification procedures. A polyelectrolyte-lipid conjugate (polyethyleneimine 1.8 kDa-dioleyl-phosphatidylinositol, PEI-PE) and polyethylene glycol 2000 (PEG) were assembled via the hypoxia-sensitive azobenzene (Azo) unit to obtain the PEG-Azo-PEI-DOPE copolymer. This copolymer can condense siRNA and shows hypoxia-induced cellular internalization and reporter gene downregulation in vitro and tumor accumulation in vivo after parenteral administration (Perche et al., Angew Chem Int Ed Engl 53:3362-3366, 2014). We also detail procedures to evaluate hypoxia-targeted polymers both in monolayer cultures, cancer cell spheroids and in tumor xenografts murine models.

Abstract B49: Cancer cell spheroids as a model to study sequential chemotherapy protocols

This study aimed at comparing six combinations of priming drug / doxorubicin (DOX) in a spheroid model. Three-dimensional cultures of drug-resistant cancer cells (spheroids) are physiologically relevant[1] and have been used as models of limited drug penetration[2]. Enhanced tumor accumulation and therapeutic efficacy of liposomal DOX after apoptosis induction by paclitaxel (PTX) has been reported[3]. Moreover, mitochondrial depolarization is correlated with clinical response[4]. Here we proposed use of spheroids to evaluate sequential chemotherapy combinations for potentiation of DOX cytotoxicity after pretreatment with cytotoxic concentrations of clinically relevant drugs. To this aim, 400–500 μm NCI-ADR-RES (ovarian carcinoma) were used. We first incubated spheroids 48h with mitoxantrone (MXO), cisplatin (CIS), dexamethasone (DXM), methotrexate (MTX), sclareol (SCL) and PTX as first line (priming) agents at concentrations inducing a 1.5- to 2.5-fold lactate dehydrogenase (LDH) release compared to untreated cells. For DOX accumulation studies, these were further cultured in the presence of 25 μM of DOX during 24h before determination of DOX content (nanomoles DOX/mg of proteins) by fluorescence. For DOX cytotoxicity evaluation, pretreated spheroids were incubated 72h with 100 μM of DOX before assessment of viability with a Cytotox 96 nonradioactive cell viability kit (Promega). Three DOX forms were used: free drug (DOX), distearyl1,2-distearoyl-sn-glycero-3-phospho-ethanolamine-N-[amino(polyethylene glycol)-2000] / DOX micelles (MDOX, 13 nm) prepared according to [5, 6], and liposomal DOX (LDOX, Lipo-Dox®, 130 nm, Sun Pharma India). The accumulation pattern of these forms was DOX = MDOX > LDOX as previously reported[7]. We document enhanced DOX, MDOX and LDOX accumulation after spheroids pretreatment with mitoxantrone (2.8-; 1.8- and 2.5-fold, respectively) and increased distribution of DOX and MDOX in PTX primed spheroids (2- and 1.4-fold, respectively). CIS, DXM, SCL and MTX failed at enhancing free, micellar or liposomal DOX distribution in spheroids. Improved DOX accumulation obtained with MXO and PTX resulted in respectively 1.4- and 2.2-fold higher LDH release compared to spheroids treated only with DOX. Interestingly, although preincubation with SCL did not promote DOX, MDOX or LDOX accumulation, LDH release of spheroids cultured with SCL before DOX treatment was 2.5-fold higher than ones treated only with DOX. While LDOX toxicity was not increased by any pretreatment, viability of cells incubated with MDOX decreased further when pretreated with PTX and MXO (1.5- and 1.8-fold compared to MDOX respectively). Low LDOX toxicity is in agreement with [7] and may be due to insufficient DOX release[8]. We report for the first time use of spheroids to test DOX toxicity potentiation as free, micellar or liposomal forms. Out of the six inducers tested, three (PTX, MXO and SCL) enhanced DOX accumulation and/or toxicity. Results suggest different mechanisms of priming for these three drugs and support sequential chemotherapy with PTX, MXO or SCL and DOX. Acknowledgments: This work was supported by grant CCNE IUCA151881 to V.P. Torchilin.