Article pubs.acs.org/molecularpharmaceutics
In Vitro Efficacy of Paclitaxel-Loaded Dual-Responsive Shell CrossLinked Polymer Nanoparticles Having Orthogonally Degradable Disulfide Cross-Linked Corona and Polyester Core Domains Sandani Samarajeewa,† Ritu Shrestha,† Mahmoud Elsabahy,†,§ Amolkumar Karwa,‡ Ang Li,† Ryan P. Zentay,† James G. Kostelc,‡ Richard B. Dorshow,*,‡,∥ and Karen L. Wooley*,† †
Department of Chemistry, Department of Chemical Engineering, and Laboratory for Synthetic-Biologic Interactions, Texas A&M University, College Station, Texas 77842, United States ‡ Covidien Pharmaceuticals R&D, Hazelwood, Missouri 63042, United States § Department of Pharmaceutics, Faculty of Pharmacy, Assiut University, Assiut, Egypt ∥ MediBeacon, LLC, St. Louis, Missouri 63108, United States S Supporting Information *
ABSTRACT: Paclitaxel-loaded shell cross-linked polymeric nanoparticles having an enzymatically and hydrolytically degradable poly(lactic acid) core and a glutathione-responsive disulfide cross-linked poly(oligoethylene glycol)-containing corona were constructed in aqueous solution and investigated for their stimuli-responsive release of the embedded therapeutics and in vitro cytotoxicity. Paclitaxel release from the nanoparticles in PBS buffer was accelerated in the presence of glutathione at both pH 5.5 and pH 7.4, reaching ca. 65% cumulative drug release after 8 d, whereas only ca. 50% and 35% extents of release were observed in the absence of glutathione at pH 5.5 and pH 7.4, respectively. Enzyme-catalyzed hydrolysis of the nanoparticle core resulted in the degradation of ca. 30% of the poly(lactic acid) core to lactic acid within 12 h, with coincidently triggered paclitaxel release of ca. 37%, as opposed to only ca. 17% release from the uncatalyzed nanoparticles at pH 7.4. While empty nanoparticles did not show any inherent cytotoxicity at the highest tested concentrations, paclitaxel-loaded nanoparticles showed IC50 values that were similar to those of free paclitaxel at 72 h incubation with KB cells and were more efficacious at ca. 3-fold lower IC50 value (0.031 μM vs 0.085 μM) at 2 h of incubation. Against human ovarian adenocarcinoma cells, the paclitaxel-loaded nanoparticles exhibited a remarkable ca. 11-fold lower IC50 than a Taxol-mimicking formulation (0.0007 μM vs 0.008 μM) at 72 h of incubation. These tunable dual-responsive degradable nanoparticles show great promise for delivery of paclitaxel to tumor tissues, given their superior in vitro efficacies compared to that of free paclitaxel and Taxol-mimicking formulations. KEYWORDS: paclitaxel, degradable, poly(DL-lactic acid), poly(DL-lactide), cell viability, disulfide cross-linker, polymeric nanoparticles
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by the FDA for administration in the United States are Taxol, PTX dissolved in Cremophor and ethanol7 in the treatment of ovarian and advanced breast cancer, and Abraxane, an albumin bound PTX-nanoparticle8 in the treatment of advanced or recurrent breast cancer. Block copolymer micelles, with their amphiphilic core−shell morphology and tunability of internal and external properties, overall shapes, and sizes, allow for improved drug solubility and have emerged over the past few decades as promising carriers for small molecule chemotherapeutics.9 For rapid and effective clinical translation, it is imperative that these micellar assemblies are composed of biocompatible materials, which can encapsulate therapeutics, gate their release, provide efficient
aclitaxel (PTX) has received significant attention since its discovery in the late 1960s1 as a highly effective drug against a wide variety of tumors including breast, ovarian, nonsmall cell lung cancers, brain tumor, and AIDS-related Kaposi’s sarcoma. However, the use of PTX as a successful chemotherapeutic agent has been limited by its inherently poor water solubility, low selectivity, and toxic side-effects. To overcome these limitations, several different formulations of PTX have been developed, and some of these are either in clinical trials or have been approved for administration by the FDA. For example, Genexol-PM is a formulation of PTX in micelles constructed from diblock copolymer poly(ethylene glycol)block-poly(D,L-lactic acid) that is in phase II and IV clinical trials against advanced pancreatic cancer and breast cancer, respectively,2,3 and NK105 is another micellar formulation constructed from poly(ethylene oxide) and modified-polyaspartate block copolymers that is currently being investigated in phase II and III clinical trials against stomach and breast cancers, respectively.4−6 Formulations that have been approved © 2013 American Chemical Society
Received: Revised: Accepted: Published: 1092
October 16, 2012 January 16, 2013 January 30, 2013 February 19, 2013 dx.doi.org/10.1021/mp3005897 | Mol. Pharmaceutics 2013, 10, 1092−1099
Molecular Pharmaceutics
Article
Figure 1. (A) Syntheses of PDLLA60 homopolymer and P(OEGMEA0.33-co-NAS0.67)66-b-PDLLA60 diblock copolymer by sequential ROP and RAFT polymerization. (B) Size exclusion chromatography traces of the homopolymer (red) and diblock copolymer (blue) samples, showing chain extension with maintenance of narrow molecular weight distribution.
Following one of our recent investigations on the incorporation of PTX into nondegradable SCK nanoparticles that showed cell-killing abilities in vitro19 and as an extension to our previous work based on investigation of the degradability of poly(L-lactic acid)-containing shell cross-linked nanoparticles,13 we have developed a block copolymer micellar system consisting of a similar hydrophobic poly(DL-lactic acid) (PDLLA) core that can encapsulate PTX and a hydrophilic shell consisting of poly(oligo(ethylene glycol) methyl ether acrylate-copolymer-N-acryloyloxysuccinimide) (P(OEGMEA-coNAS)) that has been cross-linked with a bioreducible disulfide cross-linker. The presence of OEGMEA in the shell region is expected to act as a PEG coating that would prolong the circulation time of the nanoparticles in vivo.30 The covalent cross-linking throughout the shell region provides structural stability to the nanostructure and acts as a preventive gate that minimizes the premature release of its payload, while the use of a bioreducible cross-linker takes advantage of the elevated levels of glutathione present in cells to allow for cleavage of the disulfide bond and promote triggered release of therapeutics. Moreover, the hydrolytically labile polyester core material offers additional mechanisms for promoted release. Ultimately, the orthogonal breakdown of the shell cross-links and the core chain segments was designed to lead to complete breakdown of the nanostructures.
delivery, and eventually be cleared through biological systems upon hydrolysis. Therefore polymeric micelles that constitute degradable polyester moieties within the nanoscopic framework are of particular interest and have shown great potential as vehicles for delivery of active therapeutics.10−14 Genexol-PM and NK105 are important examples of degradable block copolymer micellar packages of PTX, having polyester or polyamide block segments; however, there are opportunities for the enhancement of the compositions, structures, and properties to improve their chemotherapeutic delivery performance. Beyond the simple supramolecular assembly of block copolymers into micellar morphologies, advancements have been made on their structural stabilization by performing covalent cross-linking within the hydrophobic core domain15−17 and/or the hydrophilic shell domain18−20 to prevent the premature release of encapsulated therapeutics and dissociation of the block copolymer micelles upon infinite dilution during blood circulation when administered intravenously.9 Our group and others have explored shell crosslinked knedel-like (SCK) nanoparticles toward the design of multifunctional imaging agents,21−23 which are expected to not only prevent early disintegration of the nanostructures before reaching their target sites but also provide control over the rate of release of entrapped therapeutics.18,20 Although the majority of the conventional cross-linked nanoparticles have utilized nondegradable cross-linkers, several reversibly cleavable linkages also have been incorporated into the cross-linkers, such as reducible disulfide24,25 and pH cleavable26 and hydrolyzable ester bonds15 to further facilitate degradability of the nanoparticles and to design therapeutic delivery systems that can respond to particular stimuli, that are present either extraor intracellularly. Stimuli-responsive nanostructures have drawn attention as intelligent materials in drug delivery due their abilities to sense changes in a specific environment and rapidly stimulate structural and/or morphological responses. For example, therapeutics can be loaded into polymeric nanoconstructs, and the release of these encapsulated guest molecules can then be triggered by external stimuli such as changes in pH,20,27 light,28 and temperature.29 As the intracellular environment is known to contain elevated levels of glutathione (GSH), as opposed to the extracellular environment (ca. 10 mM vs 2 μM),25 disulfide bonds that can be cleaved by GSH have been used to construct drug, protein, and gene delivery systems.
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RESULTS AND DISCUSSION PTX-loaded bioreducible and hydrolytically degradable SCK nanoparticles, wherein the functionalities designed to undergo cleavage reactions by orthogonal reduction or hydrolysis chemistries were placed within different regions of the nanoscopic framework, were constructed by the aqueous coassembly of paclitaxel with a novel amphiphilic diblock copolymer poly(oligo(ethylene glycol) methyl ether acrylate0.33-copolymer-N-(acryloyloxy)succinimide0.67)66-block-poly(DL-lactic acid)60, P(OEGMEA0.33-co-NAS0.67)66-b-PDLLA60, followed by cross-linking between the N-hydroxysuccinimide (NHS)-activated acrylic acid (NAS) functionalities presented within the shell by the addition of cystamine, a diamine crosslinker containing a central, cleavable disulfide linkage. Our design of the amphiphilic diblock copolymer P(OEGMEA0.33co-NAS0.67)66-b-PDLLA60 precursor to the SCKs incorporates a hydrophobic, hydrolytically degradable PDLLA segment that provides an isolated cargo space for favorable partitioning of 1093
dx.doi.org/10.1021/mp3005897 | Mol. Pharmaceutics 2013, 10, 1092−1099
Molecular Pharmaceutics
Article
Scheme 1. Preparation of a SCK Nanoparticle Having Glutathione-Responsive Shell Cross-Links and a Hydrolytically Degradable Core Domain by the Co-assembly of Amphiphilic Diblock Copolymers P(OEGMEA0.33-co-NAS0.67)66-b-PDLLA60 with PTX Followed by Covalent Shell Crosslinking with Cystamine Small Molecules
PTX in its micellar form,31 surrounded by a P(OEGMEA-coNAS) copolymer segment that provides hydrophilicity and built-in functionality.32 While the NAS functionalities in the statistical copolymer segment were utilized for cross-linking, the oligo(ethylene glycol) units were expected to mimic the role of poly(ethylene glycol) (PEG) by forming a hydrophilic corona around the micelles upon self-assembly of the block copolymers, to achieve prolonged circulation in vivo.30 Sequential ring-opening polymerization (ROP) and reversible addition-fragmentation chain transfer (RAFT) polymerization were employed to obtain the initial PDLLA 60 homopolymer and subsequent P(OEGMEA0.33-co-NAS0.67)66b-PDLLA60 diblock copolymer (Figure 1A). The degrees of polymerization were determined, and well-defined structures were confirmed for the polymers by a combination of 1H NMR spectroscopy and gel permeation chromatography (GPC). GPC analyses showed monomodal molecular weight distributions with polydispersity indices (PDI) less than 1.3 (Figure 1B), indicating the controlled nature of the polymerizations. Assuming full retention of the trithiocarbonate chain end, 1H NMR spectroscopy allowed for determination of the degree of polymerization of PDLLA, by comparing the unique terminal benzyl aromatic protons resonating from 7.38 to 7.22 ppm with the broad methyl and methine proton signals of the PDLLA repeat units resonating from 1.64 to 1.38 ppm and 5.26 to 5.02 ppm, respectively. Additionally, the degree of polymerization of the P(OEGMEA-co-NAS) segment was calculated on the basis of both conversion and end group analysis from 1H NMR spectroscopy, by comparing the PDLLA methine proton signal to that of the methyl protons of OEGMEA and broad methylene protons of NAS from 3.41 to 3.31 ppm and 2.92 to 2.62 ppm, respectively. PTX-loaded SCKs and control SCKs (without PTX) were prepared from P(OEGMEA0.33-co-NAS0.67)66-b-PDLLA60 by treating the two samples to the same conditions throughout the process, with the exception of the addition of the therapeutic agent (Scheme 1). As PTX is a highly hydrophobic molecule with poor water solubility, the drug encapsulation into the SCKs was achieved by a co-assembly technique. Loading of PTX after complete production of the SCKs, by a postassembly loading technique followed in our previous work with doxorubicin-loaded SCKs,18,20,33 was unable to achieve sufficient PTX levels (
