Investigating the influence of block copolymer micelle length on cellular uptake and penetration in a multicellular tumor spheroid model.

The efficient penetration of drug nanocarriers into tumors is an important prerequisite for therapeutic and diagnostic success. The physicochemical properties of nanocarriers, including size, shape and surface chemistry have been shown to influence their transport in biological systems. Recent studies have shown that elongated nanoparticles (NPs) can exhibit advantageous properties in comparison to spherical NPs, but these experiments have involved a variety of different materials, many of which are characterized by a broad size distribution. Here we describe a series of rigid rod-like micelles of uniform width, with narrow length distributions, and common surface chemistry, and examine their cell uptake and penetration into multicellular tumor spheroids (MCTSs) formed from two human breast cancer cell lines (MDA-MB-436 and MDB-MB-231). These micelles were prepared from a polyferrocenylsilane (PFS) diblock copolymer (BCP) with a corona block consisting of a statistical polymer of aminopropyl methacrylamide and oligo(ethyleneglycol methacrylate) (PFS27-b-PAPMA3-stat-POEGMA48). The rigid rod micelles, with a common width (12 nm) and lengths ranging from 80 to 2000 nm, were prepared by seeded growth crystallization-driven self-assembly in ethanol and then transferred to water. To consider whether changing the shape of these micelles affects its uptake and penetration behavior, analogous spherical micelles were prepared by direct nanoprecipitation into water. Both micelle shape and length were found to influence cellular uptake and penetration into 500 µm MCTSs. Laser confocal fluorescence microscopy was used to examine penetration of these micelles into three-dimensional MCTS up to 90 µm depth. Micelles with an elongated shape and short length (80 nm) demonstrated the deepest penetration into the MCTSs formed by MDA-MB-436 cells. Micelles with lengths of 200 nm also showed substantial penetration into these MCTS, but the extent and depth of tumor penetration of the rod-like micelles decreased with increasing aspect ratio. MCTS of MDA-MB-231 cells had a less dense, more open structure than those formed by MDA-MB-436 cells. Here more extensive penetration was observed, particularly for the longer micelle samples.

Dual-Targeted Delivery of Nanoparticles Encapsulating Paclitaxel and Everolimus: a Novel Strategy to Overcome Breast Cancer Receptor Heterogeneity.

PURPOSE: The intratumoral heterogeneity observed in breast cancer (BC), in particular with regard to cell surface receptor expression, can hinder the success of many targeted cancer therapies. The development of novel therapeutic agents that target more than one receptor can overcome this inherent property of tumors and can facilitate their selective internalization in cancer cells. The goal of this study is to develop a drug combination-loaded nanoparticle (NP) formulation that is actively-targeted to HER2 and EGFR receptors on BC cells. METHODS: A polymeric NP formulation was prepared which co-encapsulated a synergistic combination of the chemotherapeutic agent, paclitaxel (PTX), and the mTOR inhibitor, everolimus (EVER), and is targeted to HER2 and EGFR receptors on BC cells using antibody Fab fragments as the targeting moieties. The physicochemical characteristics of the dual-targeted formulation (Dual-NP) were evaluated, along with its cytotoxic profile (in both, monolayer and 3D BC models), as well as the degree of cellular uptake in HER2high/EGFRmod and HER2neg/EGFRlow BC cells. RESULTS: Dual-NPs were found to have significantly higher cytotoxicity relative to HER2 mono-targeted (T-NPs) and untargeted NPs (UT-NPs) in HER2high/EGFRmod monolayer BC cells after 72 h exposure, while no significant difference was observed in HER2neg/EGFRlow cells. However, in the HER2high/EGFRmod spheroids, the cytotoxicity of Dual-NPs was comparable to that of T-NPs. This was thought to be attributed to the previously reported downregulation of EGFR in 3D in comparison to 2D BC models. Dual-NPs had significantly higher cellular uptake relative to UT-NPs and T-NPs in HER2high/EGFRmod BC cells after 24 h exposure, whereas in the HER2neg/EGFRlow cells, the increase in cellular uptake of the Dual-NPs was not as high as the level achieved in the HER2high/EGFRmod cells. Blocking HER2 and EGFR significantly reduced the uptake of T-NPs and Dual-NPs in the HER2high/EGFRmod BC cells, demonstrating specific binding to both EGFR and HER2. CONCLUSIONS: The dual-targeting strategy developed in this study in conjunction with a potentially promising delivery vector for a synergistic combination therapy can overcome receptor heterogeneity, yielding significant improvements in the cytotoxicity and cellular uptake in BC cells.

In Vivo Evaluation of Dual-Targeted Nanoparticles Encapsulating Paclitaxel and Everolimus.

A synergistic combination of paclitaxel (PTX) and everolimus (EVER) can allow for lower drug doses, reducing the toxicities associated with PTX, while maintaining therapeutic efficacy. Polymeric nanoparticles (NPs) of high stability provide opportunities to modify the toxicity profile of the drugs by ensuring their delivery to tumor at the synergistic ratio while limiting systemic drug exposure and the toxicities that result. The current study goal is to study the in vivo fate of human epidermal factor receptor 2 (HER2) and epidermal growth factor receptor (EGFR) dual-targeted PTX+EVER-loaded NPs (Dual-NPs) in an MDA-MB-231-H2N BC tumor-bearing mouse model. The pharmacokinetic parameters, plasma area under the curve (AUC) and half-life (t1/2), were found to be 20-fold and 3 to 4-fold higher, respectively, for the drugs when administered in the Dual-NPs in comparison to the free-drug combination (i.e., PTX+EVER) at an equivalent dose of PTX. While maintaining anti-tumor efficacy, the levels of body weight loss were significantly lower (p < 0.0001) and the overall degree of neurotoxicity was reduced with Dual-NP treatment in comparison to the free-drug combination when administered at an equivalent dose of PTX. This study suggests that Dual-NPs present a promising platform for the delivery of the PTX and EVER combination with the potential to reduce severe PTX-induced toxicities and in turn, improve quality of life for patients with BC.

Codelivery of Paclitaxel and Everolimus at the Optimal Synergistic Ratio: A Promising Solution for the Treatment of Breast Cancer.

Clinical studies examining the combination of paclitaxel (PTX) and everolimus (EVER), an mTOR inhibitor, have failed to result in significant improvements in efficacy and toxicity in patients with breast cancer (BC), relative to treatment with PTX alone. These disappointing clinical trial results have been attributed to poorly designed preclinical studies using the combination of PTX and EVER as well as the significantly different pharmacokinetic profiles of the two drugs. In the current work, the potential synergy between PTX and EVER was examined in a panel of six BC cell lines that differ in terms of their molecular subtype and drug sensitivity. Polymeric nanoparticles (NPs) were used to encapsulate PTX and EVER at an optimal synergistic ratio to achieve specific, colocalized delivery of the combination therapy in BC cell lines. Combinations of PTX and EVER (especially at relatively high doses of EVER) resulted in pronounced synergy in all BC cell lines evaluated. The optimal molar ratio of PTX:EVER was determined to be 1:0.5. The combination was delivered to BC cells at the synergistic ratio via encapsulation within polymeric NPs formed from the poly(ethylene glycol)- b-poly(lactide- co-glycolide) (PEG- b-PLGA) copolymer. The NPs had an average diameter of less than 100 nm and were capable of in vitro retention of the encapsulated PTX and EVER at the optimal synergistic molar ratio for over 7 days. Cytotoxicity data demonstrated that PTX+EVER-loaded NPs were significantly less cytotoxic than the free drug combination in MCF-7 and SKBR3 BC cell lines following 72 h, suggesting that PTX+EVER-loaded NPs remain stable and retain the drug combination loaded within the core after 72 h. The uptake of FITC-labeled NPs in SKBR3 cells was evaluated by flow cytometry, with approximately 41% of cells demonstrating detectable fluorescence after 24 h of exposure. The thorough and systematic approach used in this study to determine and evaluate a synergistic PTX:EVER ratio in conjunction with a potentially promising delivery vector for the drug combination could offer a future clinical benefit for patients with BC.

Overcoming the Road Blocks: Advancement of Block Copolymer Micelles for Cancer Therapy in the Clinic.

With countless preclinical studies on block copolymer micelles (BCMs) successfully demonstrating the superiority of these advanced drug delivery formulations over conventional formulations, it remains somehow discouraging that only a few have reached clinical evaluation and practice. With a critical eye, this review aims to compare and summarize the preclinical and clinical data available on several BCM formulations and to identify their primary role in drug delivery as "carrier" or "solubilizer". This review focuses on polymeric micelles that have reached clinical evaluation and/or are being pursued commercially. Where available, we aim to compare the pharmacokinetics, toxicity, and efficacy data obtained in preclinical studies to identify the factors that likely played a key role in a decision to move these formulations forward from the bench to a first-in-human trial. Finally, we summarize clinical data obtained to date, where available, and conclude with the impact that each formulation has had on patients in terms of safety and efficacy.

Anionic Polymerization of an Amphiphilic Copolymer for Preparation of Block Copolymer Micelles Stabilized by π-π Stacking Interactions.

In this study, an amphiphilic copolymer that includes a core-forming block with phenyl groups was synthesized by living anionic polymerization of phenyl glycidyl ether (PheGE) on methoxy-polyethylene glycol (mPEG-b-PPheGE). Characterization of the copolymer revealed a narrow molecular distribution (PDI < 1.03) and confirmed the degree of polymerization of mPEG122-b-(PheGE)15. The critical micelle concentration of the copolymer was evaluated using an established fluorescence method with the aggregation behavior evaluated by dynamic light scattering and transmission electronic microscopy. The potential of the copolymer for use in drug delivery applications was evaluated in a preliminary manner including in vitro biocompatibility, loading and release of the hydrophobic anti-cancer drug doxorubicin (DOX). A stable micelle formulation of DOX was prepared with drug loading levels up to 14% (wt%), drug loading efficiencies > 60% (w/w) and sustained release of drug over 4 days under physiologically relevant conditions (acidic and neutral pH, presence of albumin). The high drug loading level and sustained release is attributed to stabilizing π-π interactions between DOX and the core-forming block of the micelles.