ABSTRACT
The preparation, release and in vitro cytotoxicity of a novel polymeric micellar formulation of paclitaxel (PTX) were investigated. The micelles consisted of an AB block copolymer of poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol) (pHPMAmDL-b-PEG). Taking advantage of the thermosensitivity of pHPMAmDL-b-PEG, the loading was done by simply mixing of a small volume of a concentrated PTX solution in ethanol and an aqueous polymer solution and subsequent heating of the resulting solution above the critical micelle temperature of the polymer. PTX could be almost quantitatively loaded in the micelles up to 2 mg/mL. By dynamic light scattering and cryo-transmission electron microscopy, it was shown that PTX-loaded micelles have a mean size around 60 nm with narrow size distribution. At pH 8.8 and 37 degrees C, PTX-loaded micelles destabilized within 10 h due to the hydrolysis of the lactic acid side group of the pHPMAmDL. Because the hydrolysis of the lactic acid side groups is first order in hydroxyl ion concentration, the micelles were stable for about 200 h at physiological conditions. The presence of serum proteins did not have an adverse effect on the stability of the micelles during at least 15 h. Interestingly, the dissolution kinetics of pHPMAmDL-b-PEG micelles was retarded by incorporation of PTX, indicating a strong interaction between PTX and the pHPMAmDL block. The PTX-loaded micelles showed a release of the incorporated 70% of PTX during 20 h at 37 degrees C and at pH 7.4. PTX-loaded pHPMAmDL-b-PEG micelles showed comparable in vitro cytotoxicity against B16F10 cells compared to the Taxol standard formulation containing Cremophor EL, while pHPMAmDL-b-PEG micelles without PTX were far less toxic than the Cremophor EL vehicle. Confocal laser-scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS) analysis of fluorescently labelled micelles showed that pHPMAmDL-b-PEG micelles were internalized by the B16F10 cells. The present results suggest that pHPMAmDL-b-PEG block copolymer micelles are a promising delivery system for the parenteral administration of PTX.
Subject(s)
Antineoplastic Agents, Phytogenic/administration & dosage , Biocompatible Materials/chemistry , Drug Carriers/chemistry , Paclitaxel/administration & dosage , Animals , Antineoplastic Agents, Phytogenic/chemistry , Antineoplastic Agents, Phytogenic/pharmacology , Cell Line, Tumor , Cell Survival/drug effects , Drug Compounding , Drug Stability , Lactic Acid/chemistry , Mice , Micelles , Paclitaxel/chemistry , Paclitaxel/pharmacology , Particle Size , Polyethylene Glycols/chemistry , Polymethacrylic Acids/chemistry , Solubility , TemperatureABSTRACT
Amphiphilic AB block copolymers consisting of thermosensitive poly(N-(2-hydroxypropyl) methacrylamide lactate) and poly(ethylene glycol), pHPMAmDL-b-PEG, were synthesized via a macroinitiator route. Dynamic light scattering measurements showed that these block copolymers form polymeric micelles in water with a size of around 50 nm by heating of an aqueous polymer solution from below to above the critical micelle temperature (cmt). The critical micelle concentration as well as the cmt decreased with increasing pHPMAmDL block lengths, which can be attributed to the greater hydrophobicity of the thermosensitive block with increasing molecular weight. Cryogenic transmission electron microscopy analysis revealed that the micelles have a spherical shape with a narrow size distribution. 1H NMR measurements in D2O showed that the intensity of the peaks of the protons from the pHPMAmDL block significantly decreased above the cmt, indicating that the thermosensitive blocks indeed form the solidlike core of the micelles. Static light scattering measurements demonstrated that pHPMAmDL-b-PEG micelles with relatively large pHPMAmDL blocks possess a highly packed core that is stabilized by a dense layer of swollen PEG chains. FT-IR analysis indicated that dehydration of amide bonds in the pHPMAmDL block occurs when the polymer dissolved in water is heated from below to above its cmt. The micelles were stable when an aqueous solution of micelles was incubated at 37 degrees C and at pH 5.0, where the hydrolysis rate of lactate side groups is minimized. On the other hand, at pH 9.0, where hydrolysis of the lactic acid side groups occurs, the micelles started to swell after 1.5 h of incubation and complete dissolution of micelles was observed after 4 h as a result of hydrophilization of the thermosensitive block. Fluorescence spectroscopy measurements with pyrene loaded in the hydrophobic core of the micelles showed that when these micelles were incubated at pH 8.6 and at 37 degrees C the microenvironment of pyrene became increasingly hydrated in time during this swelling phase. The results demonstrate the potential applicability of pHPMAmDL-b-PEG block copolymer micelles for the controlled delivery of hydrophobic drugs.
Subject(s)
Micelles , Polymers/chemistry , Temperature , Acrylamides/chemical synthesis , Acrylamides/chemistry , Chemical Phenomena , Chemistry, Physical , Lactates/chemical synthesis , Lactates/chemistry , Molecular Structure , Particle Size , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/chemistry , Polymers/chemical synthesis , Polymethacrylic Acids/chemical synthesis , Polymethacrylic Acids/chemistry , Surface Properties , Time FactorsABSTRACT
A novel class of thermosensitive and biodegradable polymers, poly(N-(2-hydroxypropyl) methacrylamide mono/di lactate) (poly(HPMAm-mono/di lactate)), was synthesized. The cloud points (CP) of poly(HPMAm-monolactate) and poly(HPMAm-dilactate) in water were 65 and 13 degrees C, respectively. The lower CP for poly(HPMAm-dilactate) is likely due the greater hydrophobicity of the dilactate side group over the monolactate side group. The CP of poly(HPMAm-monolactate-co-HPMAm-dilactate) increased linearly with mol % of HPMA-monolactate, which demonstrates that the CP is tuneable by the copolymer composition.
