Article pubs.acs.org/Biomac
Decreased Core Crystallinity Facilitated Drug Loading in Polymeric Micelles without Affecting Their Biological Performances Jingxin Gou, Shuangshuang Feng, Helin Xu, Guihua Fang, Yanhui Chao, Yu Zhang, Hui Xu,* and Xing Tang* School of Pharmacy, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang 110016, China
Downloaded by FLORIDA STATE UNIV on September 8, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.biomac.5b00826
S Supporting Information *
ABSTRACT: Cargo-loading capacity of polymeric micelles could be improved by reducing the core crystallinity and the improvement in the amount of loaded cargo was cargo-polymer affinity dependent. The effect of medium chain triglyceride (MCT) in inhibiting PCL crystallization was confirmed by DSC and polarized microscope. When incorporating MCT into polymeric micelles, the maximum drug loading of disulfiram (DSF), cabazitaxel (CTX), and TM-2 (a taxane derivative) increased from 2.61 ± 0.100%, 13.5 ± 0.316%, and 20.9 ± 1.57% to 8.34 ± 0.197%, 21.7 ± 0.951%, and 28.0 ± 1.47%, respectively. Moreover, the prepared oil-containing micelles (OCMs) showed well-controlled particle size, good stability, and decreased drug release rate. MCT incorporation showed little influence on the performances of micelles in cell studies or pharmacokinetics. These results indicated that MCT incorporation could be a core construction module applied in the delivery of hydrophobic drugs. chemical conjugation of small molecular pendants15−17 or physical blending of different polymers18,19 have been reported as having the ability to improve drug loading. In the case of micelles composed of polymers that are semicrystalline, such as polycaprolactone (PCL), micelle core crystallinity is another factor influencing drug loading capacity of the polymer: increased crystallinity would lead to a reduced drug loading capacity due to the tight packing of the polymer chains makes it hard for drug molecules to be incorporated into the crystal region.20 So, it is possible to improve drug loading by reducing core crystallinity. Lowering the core crystallinity is currently achieved by changing the chemical composition of the polymers through introduction of different monomers, thus, synthesized random copolymers that disrupted the ordered structure of the hydrophobic block20 or preparing micelles composed of stereoblock copolymers that reduce core crystallinity by stereocomplex formation.21 It is hypothesized that the incorporation of small-molecular hydrophobic substances might inhibit polymer crystallization in the micelle core as they are able to infiltrate between polymer chains to disturb their packing, hence, improve drug loading through expanded amorphous region. However, for the inhibitors employed to reduce the core crystallinity of micelles subjected to medical uses, they need to be inert and nontoxic. Here we report the crystallization-inhibiting effect of medium chain triglycerides (MCT) on micelles composed of mPEG-bPCL simply by addition of MCT into micelle cores. Medium
1. INTRODUCTION The core−shell structure of polymeric micelles provides them with the ability to prolong blood circulation and solubilize substances that are barely soluble in water. Using this property, polymeric micelles have been studied as antitumor drug delivery systems for decades. In addition, to effectively deliver drugs to their targets, methods for block copolymer functionalization,1,2 or shape alteration of nanoassemblies3,4 have been developed. Another important aspect in the construction of drug delivery systems is drug loading. Currently there are methods reported to load some drugs through covalent bonding, coordination bonding, or noncovalent interactions, including electrostatic complex formation or hydrogen bonding.5−10 However, fewer studies were focused on the optimization in loading of hydrophobic drugs with no “functionalizable” groups. Most hydrophobic drugs are encapsulated into micelle cores primarily through a hydrophobic interaction and hydrogen-bond formation,11 and the drug loading is left to be determined by the polymer-drug compatibility. However, due to huge variation in drug structure, the loading of different drugs is very structuredependent, so the core-forming blocks we now have in hand may not be able to meet the delivery requirements of all drugs.12 This indicates that a “drug loading-oriented carrier design” pattern is required for constructing drug delivery systems. “Drug loading-oriented carrier design”, which focuses on the hydrophobic core of the micelle, involves optimized loading of drugs with different physical-chemical properties. Calculating Flory−Huggins interaction parameters (χsp) of polymers and drugs is a classic way to find a suitable carrier for a particular drug and vice versa.13,14 More recently, other methods aimed at altering the properties of the hydrophobic core, including © XXXX American Chemical Society
Received: June 21, 2015 Revised: August 27, 2015
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DOI: 10.1021/acs.biomac.5b00826 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
min, then cooled to 30 °C, and finally heated to 80 °C. The melting temperature and melting enthalpy were recorded. Crystallization of PCL samples with/without MCT was monitored by polarizing microscopy. Solutions of PCL with different MCT contents (0, 20, and 40%) were prepared in a similar way to the DSC measurements, except that 2−3 drops of the solutions were added to clean glass slides at room temperature in a fume hood and allowed to form thin films. AFM measurement was conducted using Cypher S (Asylum Research). All the images were obtained under tapping mode and the scan rate was 4.88 Hz. 2.3. Preparation of OCMs Containing Different Amounts of MCT. OCMs containing different amounts of MCT were prepared by dialysis. Briefly, polymer (40 mg) was mixed with predetermined amounts of MCT (5, 10, 20, 40, 60, and 80% of the weight of polymer used for all the three polymers; 120% for mPEG5k-b-PCL13k and 150% for mPEG5k-b-PCL16k) and then dissolved in THF (2 mL). Purified water (4 mL) was added dropwise into the solution under stirring. The resultant mixture was then transferred to a dialysis bag (MWCO: 3500) and dialyzed against deionized water for 24 h. The sample was then centrifuged at 10000 rpm for 10 min after dialysis to remove precipitates, if any. 2.4. Characterization of Micelles. The particle size and distribution of MCT-free micelles composed of mPEG5k-b-PCL10k, mPEG5k-b-PCL13k, and mPEG5k-b-PCL16k as well as the micelles containing different amounts of MCT composed of those three polymers were measured by dynamic light scattering (DLS) with a NICOMP 380 Submicron Particle Sizer (Particle Sizing System, Santa Barbara, CA) at room temperature. AFM was used to characterize micelles with/without MCT. The morphology of the MCT-free micelles and OCMs were observed using a JEM-2100 transmission electronic microscope (JEOL) with 200 kV acceleration voltage. Samples were stained with phosphotungustic acid. The detailed morphology of OCM with high MCT content (80%) was observed using cryogenic-TEM (FEI, Tecnai 20, 200 kV). Critical aggregation concentration (CAC) of all micelles and OCMs were determined using pyrene by recording the excitation spectra from 300 to 350 nm by fixing the emission wavelength at 390 nm. Core polarity of all the micelles and OCMs was determined using pyrene fluorescence.18 The emission spectrum of pyrene for each sample in the range from 360 to 430 nm was recorded using an F-7000 spectrophotometer (Hitachi) at an excitation wavelength of 335 nm. The ratio of the fluorescent intensity of the first peak to the third peak in the emission spectrum was used to reflect the core polarity. 1,3-(1,1′Dipyrenyl)-propane (P3P) was used for the determination of the core microviscosity of both MCT-free micelles and OCMs by measuring the ratios of the fluorescence intensities of the monomer and excimer (IM/ IE) at 376 and 480 nm, respectively. The emission spectra of P3P were recorded on an F-7000 spectrophotometer (Hitachi) at an excitation wavelength of 333 nm. The scanning speed of both experiments was 240 nm/min, and the bandwidth slits were set at 2.5 nm. 2.6. Drug Loading and Stability of OCMs. The maximum drug loading capacity of MCT-free micelles and OCMs (MCT content: 5, 10, 25, and 50% of the weight of the polymers) composed of mPEG5k-bPCL10k was determined by drug addition in excess during micelle preparation. Samples were prepared by dialysis as described previously. The concentrations of the drugs in MCT-free micelles or OCMs were measured by HPLC. Drug-loaded MCT-free micelles and OCMs (MCT content: 50% of the weight of the polymers) composed of mPEG5k-b-PCL10k were stored at room temperature. The particle size and drug concentration in micelles and OCMs were measured on days 0, 1, 2 and 3. 2.7. In Vitro Release Studies. The release profiles of MCT-free micelles and OCMs (MCT content: 40 and 80% of the weight of the polymers) composed of mPEG5k-b-PCL10k were investigated by dialysis diffusion method. The drug to excipient ratio in both MCT-free micelles and OCMs was fixed at 0.025. 2.8. In Vitro Cytotoxicity and Cellular Uptake Studies. Cytotoxicity of the empty OCMs with a 50% MCT content and CTX-loaded, MCT-free micelles or OCMs was assayed by MTT
chain triglyceride (MCT) is a pharmaceutical adjuvant widely used in the preparation of lipid-based formulations, including emulsions, nanocapsules, and lipid nanoparticles. As a component rich in horse milk fats,22 MCT is more biocompatible and safer than many other probable hydrophobic crystallization inhibitors. Moreover, it can act as a solvent for extremely lipophilic drugs,23 and according to the results of our previous studies on lipid emulsions, MCT showed certain solubility to drugs like cabazitaxel (CTX),24 TM-2 (a taxane derivative),25 and disulfiram (DSF).26 Our results indicated that the MCT incorporated in micelle cores acted as a crystallization inhibitor and core expander, and the drug loading of CTX, TM-2, and DSF in such oil containing micelles (OCMs) was improved significantly (Scheme 1).
Downloaded by FLORIDA STATE UNIV on September 8, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.biomac.5b00826
Scheme 1. Schematic Illustration of the Relationship between Core Crystallinity and Drug Loading Capability
2. EXPERIMENTAL SECTION 2.1. Materials. mPEG5k-OH (Aladdin Agent Co., Shanghai, P.R. China) was dried under vacuum at 80 °C before use. ε-Caprolactone (εCL, Aladdin Agent Co., Shanghai, P.R. China) and n-butanol (Aladdin Agent Co., Shanghai, P.R. China) were dried over CaH2 and phosphorus pentoxide, respectively, and distilled under reduced pressure. Stannous octotate (National Medicine Chemical Reagent Ltd. Co., Shanghai, P.R. China) was used as received. MCT was obtained from Lipoid KG (Ludwigshafen, Germany). Cabazitaxel (CTX) was a gift from the Medicinal Chemistry Lab of Yantai University. TM-2 was provided by Fudan University. Disulfiram (DSF) was purchased from Jinan Ruijing Pharmaceutical Co. Ltd. (Jinan, Chian). Dialysis bags (MWCO: 3.5k Da and 14k Da) were purchased from Ruidahenghui Co., Ltd. Beijing. All other agents used were analytical grade. 2.2. Measurements. DSC measurements were performed using a DSC 1 differential scanning calorimeter (Mettler Toledo, Switzerland) equipped with a refrigerated cooling system. Samples (approximately 2 mg, accurately weighed) were placed in hermetically sealed aluminum pans with pin holes made on the lids to allow escape of moisture. Nitrogen was used as a purge gas at a flow rate of 80 mL/min, and the samples were first heated from room temperature to 80 °C, kept for 5 B
DOI: 10.1021/acs.biomac.5b00826 Biomacromolecules XXXX, XXX, XXX−XXX
Article
Biomacromolecules
Downloaded by FLORIDA STATE UNIV on September 8, 2015 | http://pubs.acs.org Publication Date (Web): September 2, 2015 | doi: 10.1021/acs.biomac.5b00826
method with CTX dissolved in DMSO as a positive control. Cell uptake of OCMs was studied using coumarin-6 as a fluorescence probe. 2.9. Pharmacokinetic Studies of CTX Solutions, CTX-Loaded, MCT-Free Micelles, and OCMs. This experiment was approved by the Animal Ethics Committee of Shenyang Pharmaceutical University. The method of UPLC-MS/MS analysis employed in this study was similar to the one we reported previously.24 The pharmacokinetic profiles of CTXsolutions, CTX loaded MCT-free micelles and OCMs (MCT content: 50%) were evaluated in Sprague−Dawley rats. Samples were quantified using a calibration curve over the range of 10−2000 ng/mL and the correlation coefficient was higher than 0.99. 2.10. Statistical Analysis. The pharmacokinetic parameters of CTX in rats were calculated using DAS 2.1 supplied by the Pharmacological Society of China (Beijing, China). All data are presented as mean ± SD and analyzed using Student’s t test. Statistical significance was determined as a P value of
