Article pubs.acs.org/bc
Synthetic Modification of Carboxymethylcellulose and Use Thereof to Prepare a Nanoparticle Forming Conjugate of Docetaxel for Enhanced Cytotoxicity against Cancer Cells Mark J. Ernsting, Wei-Lun Tang, Noah MacCallum, and Shyh-Dar Li* Drug Delivery and Formulation Group, Medicinal Chemistry Platform, Ontario Institute for Cancer Research, 101 College Street, Suite 800, Toronto, Ontario, M5G 0A3, Canada ABSTRACT: A nanoparticle formulation of docetaxel (DTX) was designed to address the strengths and limitations of current taxane delivery systems: PEGylation, high drug conjugation efficiency (>30 wt %), a slow-release mechanism, and a well-defined and stable nanoparticle identity were identified as critical design parameters. The polymer conjugate was synthesized with carboxymethylcellulose (CMC), an established pharmaceutical excipient characterized by a high density of carboxylate groups permitting increased conjugation of a drug. CMC was chemically modified through acetylation to eliminate its gelling properties and to improve solvent solubility, enabling high yield and reproducible conjugation of DTX and poly(ethylene glycol) (PEG). The optimal conjugate formulation (Cellax) contained 37.1 ± 1.5 wt % DTX and 4.7 ± 0.8 wt % PEG, exhibited a low critical aggregation concentration of 0.6 μg/mL, and formed 118−134 nm spherical nanoparticles stable against dilution. Conjugate compositions with a DTX degree of substitution (DS) outside the 12.3−20.8 mol % range failed to form discrete nanoparticles, emphasizing the importance of hydrophobic and hydrophilic balance in molecular design. Cellax nanoparticles released DTX in serum with near zero order kinetics (100% in 3 weeks), was internalized in murine and human cancer cells, and induced significantly higher toxic effects against a panel of tumor cell lines (2- to 40-fold lower IC50 values) compared to free DTX.
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develop polymer therapeutics, and has also given rise to nanomedicine, where structures comprising liposomes,19−21 polymeric micelles, 22−24 polymersomes, 25−27 and dendrimers28−30 can effectively deliver drug through this passive targeting effect.31 However, polymers and particles can be readily opsonized and cleared in the reticulo-endothelial system (RES, bone marrow, liver, and spleen), reducing the effectiveness of these delivery systems.19,32 PEGylation of a polymer or nanoparticle can reduce interaction of opsonins with the underlying chemistry through steric hindrance and reduction of hydrophobic and electrostatic interactions, minimizing clearance of the particle in the RES.19,33−36 Hydrophobic drugs such as PTX can be loaded noncovalently in nanoparticle formulations. Notable within this field are the PEG-PLA micelles (Genexol, now in phase II clinical trials)37 and NK105, a PEG-aspartic acid formulation.23 Both formulations improve upon administration of PTX through elimination of the Cremophor-based delivery vehicle, which causes hypersensitivity issues in human patients,19 and by enabling treatment with an increased dose to improve efficacy. In comparison to Genexol and NK105, Opaxio (also known as Xyotax and Poliglumex) is a PTX−polyglutamate conjugate.
INTRODUCTION The taxanes paclitaxel (PTX) and docetaxel (DTX) are common cancer therapy agents with a broad spectrum of antitumor activity, and are currently formulated with Cremophor EL/ethanol/saline1 and Tween80/ethanol/saline, respectively, due to their water insolubility, each of which causes hypersensitivity reactions, requiring premedication regimes.2,3 DTX is replacing PTX in clinical applications due to enhanced action,4 and reports on polymeric formulations of DTX are indicating that the drug can be more effectively and safely delivered.5−7 The use of polymers to improve solubility, pharmacokinetics (PK), pharmacodynamics (PD), bioavailability, efficacy, and toxicity profiles is commonly defined as polymeric therapeutics, and is a thriving field of study.8−10 It has been demonstrated that soluble macromolecules with molecular weights (MW) above 40 kDa circulate longer than small molecule drugs in the bloodstream due to reduced renal clearance.11,12 Further, Matsumura and Maeda observed that tumor vasculature is highly permeable, and that high MW polymers or nanoparticles can selectively accumulate in these tissues as a result.13 Neovascular structures in malignant tissues tend to contain gaps through which 50 mol %) and oxidized CMC, and tested the effects of these polymers on s.c. sarcoma tumors in rat models, and reported moderate antitumor effect from a single i.p. injection.50 Auzenne et al. conjugated PTX to HA (DS 10 mol %), and performed in vitro and in vivo efficacy assays.51 Mice were implanted with ovarian carcinoma xenografts in the peritoneal cavity, and were treated with an intraperitoneal injection of 200 mg/kg PTX-HA, a treatment which effectively cured the mice and was well tolerated. Inoue et al. reported on a camptothecin analogue (DX-8591) conjugated to a carboxymethyldextran via a peptide spacer (DS 40 mol %), which demonstrated strong action against tumors in mice models and human patients.52,53 Wang et al. conjugated PTX to heparin (20 wt %), but could only form nanostructures when coformulated with free PTX. 54 In short, polysaccharides appear to have potential as polymer therapeutics, with positive outcomes being reported for selected compositions. However, polysaccharides are generally watersoluble and are incompatible with organic solvents, limiting drug conjugation chemistry, resulting in low coupling yields for hydrophobic drugs (30 mol % feed or use of higher MW PEG5000) disrupted particle forming properties. For effective nanoparticle forming properties, the hydrophobic (DTX) and hydrophilic (PEG) elements of the Cellax macromolecule were balanced, so that when these amphiphilic structures contacted isotonic aqueous solution, they would assemble into stable nanoparticles and protect the drug cargo from direct exposure to serum enzymes. Particle Formulation and Stability. Nanoparticles were prepared using the well-established nanoprecipitation method67 by slow addition of a MeCN or THF solution of Cellax polymer conjugate into aqueous media (10× dilution), and desired particles were produced provided the concentration of Cellax in the organic solvent solution ranged between 10 and 25 mg/mL. It was observed that particles formed from 25 mg/ mL solutions were approximately 150 nm in size, and particles formed from 10 mg/mL solutions were smaller (105−120 nm). For all subsequent work, the 10 mg/mL MeCN polymer solution and 10× dilution parameters (1 mg/mL polymer in particle solution) were set so as to provide for the smallestsized population of nanoparticles. As described in Table 1,
Figure 4. TEM analysis. Cellax particles were applied to Formvar coated copper grids, and analyzed by dark-field TEM (500 000× magnification). Six randomly picked particles were imaged, and the size was measured: particle diameter = 104 ± 15 nm.
Figure 5. Dilution stability. Cellax particles were diluted from 47 mg/ mL to 0.1 μg/mL (below the critical aggregation concentration and at the limits of particle size detection) and were stored for 24 h prior to analysis. Particle diameter increased slightly as the particles were diluted.
Table 1. Cellax Particle Size Characteristics in Different Aqueous Systems (Zetasizer Measurements) solution
size (nm)
PDI
PBS Saline (0.9%) Sucrose (10%)
134.0 ± 3.4 118.2 ± 1.8 128.5 ± 3.2
0.1 ± 0.08 0.08 ± 0.02 0.10 ± 0.04
4 mg DTX/mL) would be diluted in ∼2 mL blood volume to 2 mg Cellax/mL. Considering the scenario when 90% of particles are out of circulation, the concentration of Cellax would be still well above CAC, and the DTX cargo would remain protected from serum esterases. In addition to stability against dilution, Cellax particles were stable in storage (>2 months, ongoing study) in saline, in contrast to reports on Abraxane and various PTX micelle formulations which exhibit limited stability in suspension.73,74 In Vitro DTX Release. The release of DTX from Cellax nanoparticles in serum was analyzed to affirm that hydrolysis of the ester bonds would occur. By LC/MS analysis, two identities with an ES + MS value of 808.8 m/z were detected, corresponding to DTX and a DTX isomer, 7-epidocetaxel: these peaks were analyzed to generate a total taxane release value.57,75,76 The isomerization of Taxol (PTX) and Taxotere (DTX) in serum and the decreased activity are welldocumented metabolic phenomena,57,77,78 partly due to the unavoidable metabolism of taxanes in biological systems which supports the need to create a protective formulation for taxanes (i.e., nanoparticle encapsulated). As shown in Figure 6, the release of DTX over the course of 21 days (3.6%/day) was sustained, culminating in full release, with half of the total taxane released as active DTX. This data was contextualized
Cellax nanoparticles could be formed in a variety of isotonic solutions with defined diameter (118−134 nm, PDI < 0.1). TEM analysis (Figure 4) of the particles in normal saline (104 ± 15 nm) supported the Zetasizer measurement (118 nm ±1.5 nm). The critical aggregation concentration (CAC), determined by the DPH assay, was 0.6 μg/mL. In comparison, hyaluronic−paclitaxel conjugates have a reported critical micelle concentration (cmc) of 7.8 μg/mL,68 poly(glutamylgultaminePTX) has a cmc of 25 μg/mL,69 and PEG-polyesters typically have cmcs of the same order of magnitude (μg/mL levels) as Cellax.70−72 Cellax particles were diluted serially to 0.5 μg/mL and examined for size stability: as dilution occurred, particle size exhibited only a minor increase when diluted to the limits of Zetasizer particle size detection, and did not disassemble (Figure 5). As Cellax was stable at the CAC, it is probable that these particles will remain stable at high dilution in biological systems. For example, in a 20 g mouse model treated at 40 mg DTX/kg, 200 μL of Cellax administered i.v. (10 mg Cellax/mL, 2481
dx.doi.org/10.1021/bc200284b | Bioconjugate Chem. 2011, 22, 2474−2486
Bioconjugate Chemistry
Article
with respect to published reports on taxane conjugates. For example, release of PTX from water-soluble carboxylmethyl dextran conjugates (5.5−6.5 wt % PTX) is rapid, with total release occurring within 3−4 days.55 Release of DTX from water-soluble albumin conjugates (1.5 wt % DTX) is likewise rapid, with 40% release in 1 day.7 Release of DTX from PEG conjugates (26 wt % DTX) is more controlled, with full release in 6 days.6 Release of PTX from polyglutamic acid conjugates (35.8 wt % PTX) is slow and similar to Cellax, with 15−30% release recorded in 4−5 days.43,79 It is posited that ready access of hydrolytic enzymes to the ester bonds linking the taxane to the macromolecule may drive the high rate of hydrolysis in low DS (
