بهره برداری از مسیر درون سلولی موجود دیفرانسیلی در بافت سالم و تومور برای هدف قرار دادن گزینشی عوامل شیمی درمانی نانوذرات

Polymeric micelles with cross-linked ionic cores of poly(methacrylic acid) and nonionic shell of poly(ethylene oxide) (cl-micelles) are shown here to readily internalize in epithelial cancer cells but not in normal epithelial cells that form tight junctions (TJ). The internalization of such cl-micelles in the cancer cells proceeded mainly through caveolae-mediated endocytosis. In confluent normal epithelial cells this endocytosis route was absent at the apical side and the cl-micelles sequestered in TJ regions of the cell membrane without entering the cells for at least 24 h. Disruption of the TJ by calcium deprivation resulted in redistribution of cl-micelles inside the cells. In cancer cells following initial cellular entry the cl-micelles bypassed the early endosomes and reached the lysosomes within 30 min. This allowed designing cl-micelles with cytotoxic drug, doxorubicin, linked via pH-sensitive hydrazone bonds, which were cleaved in the acidic environment of lysosomes resulting in accumulation of the drug in the nucleus after 5 h. Such pH-sensitive cl-micelles displayed selective toxicity to cancer cells but were non-toxic to normal epithelial cells. In conclusion, we describe major difference in interactions of cl-micelles with cancer and normal cells that can lead to development of novel drug delivery system with reduced side effects and higher efficacy in cancer chemotherapy.

Recent years saw rapid emergence of polymeric nanoparticulate materials (NMs) for delivery of drugs, genes and imaging agents [1], [2], [3], [4] and [5]. Examples of materials under development include liposomes, polymer-drug conjugates, nanogels, polymeric micelles, and the like. The precise delivery of these materials to the sites of the disease, such as tumor cells, is central for successful therapies. For example, passive targeting of nanoparticles, also termed as “Enhanced Permeation and Retention (EPR)” effect, has attracted great attention in delivery of drugs and imaging agents [6] and [7]. EPR stems from an intrinsic property of certain tumors to accumulate NMs due to leaky vasculature and poor lymphatic drainage. The mechanisms of subsequent entry of NMs into target cells have also attracted great attention. The size, shape, charge and aggregation state of NMs were most recently revealed as critical determinants for their cellular entry and sub-cellular targeting [8] and [9]. Specifically, different NMs employ different endocytosis mechanisms to gain cellular entry. These mechanisms are classified as clathrin dependent (clathrin-mediated endocytosis) and clathrin independent pathways (caveolae-mediated endocytosis, clathrin- and caveolae-independent pathways and macropinocytosis) [9]. After exploitation of these entry pathways, NMs are processed through complex sorting mechanisms and are driven to specific intracellular compartments. In the field of drug delivery the mechanisms of cellular entry of NMs and their final sub-cellular distribution, could greatly affect the performance of the drugs. Therefore, understanding these mechanisms is of significance. The purpose of this work was to evaluate cellular entry of core-cross linked polymeric micelles (cl-micelles) of poly(ethylene oxide)-b-poly(methacrylic acid) (PEO-b-PMA) copolymer, that were recently proposed for delivery of anticancer drugs [10]. In an aqueous environment such micelles behave as nanoscale ionic gels, capable of swelling and changing charge in response to environmental changes (pH or ionic strength). Unexpectedly we found here that such micelles in confluent epithelial cells selectively bind with the tight junctions (TJ) and do not enter the cells. However, disruption of the TJ abolishes such localization and promotes entry of the micelles into the cells. In cancer cells with absent or dysfunctional TJ the cellular entry of such micelles is not restricted by the level of cell confluency. The micelles enter the cells selectively through caveolae-mediated endocytosis, bypass early endosomes and reach lysosomes. This suggests that observed control of the delivery of the synthetic NM through formation of TJ in normal epithelial cells, and enhanced entry into cancer cells and can be exploited for design of cancer-specific drug carriers.

This study describes cl-micelles that display a selectivity of cellular entry via caveolae-mediated endocytosis. We also show that the entry of such cl-micelles is inhibited in TJ-forming epithelial cells but permitted in cancer cells that do not form TJ. This difference is due to lack of caveolae-mediated endocytosis at the apical side of TJ-forming epithelial cells. In cancer cells, following initial stages of cellular entry cl-micelles avoid early endosomes but are ultimately routed to lysosomes. A possibility of creating cl-micelles containing a drug linked to the micelles via pH-sensitive bond, which is stable at extracellular pH but cleavable in lysosomes, is further demonstrated. Such pH-sensitive drug loaded cl-micelles can release drug in lysosomes compartments and exhibit selective toxicity to cancer cells but are not toxic to normal epithelial cells that form TJ. Overall this work demonstrates that cellular trafficking of selected NMs can be very different in cancer and normal cells and reinforces the need of investigation of peculiarities of such interactions for design of safe and efficient nanomedicines.