Particle Size and Temperature Effect on the Physical Stability of PLGA Nanospheres and Microspheres Containing Bodipy

Particle Size and Temperature Effect on the Physical Stability of PLGA Nanospheres and Microspheres Containing Bodipy
By: Sinjan De,1 and Dennis H. Robinson2 - 1Ohio Northern University, College of Pharmacy, 525 South Main Street, Ada, OH 45810
2Department of Pharmaceutical Sciences, 986025 Nebraska Medical Center, Omaha, NE 68198-6025
Submitted: July 12, 2004; Accepted: September 13, 2004
AAPS PharmSciTech
Introduction
Nanospheres and microspheres have been extensively used to deliver a wide range of drugs as they can protect the drug from metabolizing enzymes, sustain the release, be administered orally or injected locally, and target specific tissues by incorporating surface ligand moieties.1-8 A common polymer used in the formulation of nanospheres and microspheres is the biodegradable, biocompatible polymer, polylactic-co-glycolic acid (PLGA).5-9 This research is a part of the overall study of the effect of the mean diameter of the particulate delivery systems on the cellular accumulation, cytotoxicity, and efficacy of paclitaxel. During these studies, there was evidence that the temperature of storage is important in maintaining the physical integrity of particulate delivery systems.
There have been numerous publications on the rate and extent of chemical degradation of PLGA used in various drug delivery systems, including the factors that influence the kinetics of this reaction.9,10 The factors that influence the chemical degradation of PLGA are well known and include polymer molecular weight, ratio of lactic to glycolic acid in the co-polymers, polymer-drug ratio, environmental temperature, pH, and geometry of the delivery system.11 PLGA undergoes random hydrolysis to oligomers, then ultimately to the nontoxic, monomeric units, lactic and glycolic acids. Oligomers hydrolyze more rapidly than the higher molecular weight parent polymer due to the presence of more terminal carboxylic acid groups per unit molecular mass.9,12-19 Lactic and glycolic acids occur naturally in the human body and are easily eliminated through the glycolytic pathway as carbon dioxide and water. It has been shown that PLGA nanospheres and microspheres have a shelf-life of more than 3 months (PLGA 50:50, 0.63 dL/g).5 However, to our knowledge, the influence of storage temperature, duration of storage, and particle size on the physical stability and morphology of nanospheres and microspheres has not been reported.
During preparation, PLGA particulate delivery systems are lyophilized and, although compactly arranged, can be readily resuspended in aqueous media after being stored at 4?C. However, we observed that particle size and storage temperature are important in maintaining the integrity of these delivery systems. Therefore, the purpose of this research was to study the effect of storage temperature, storage duration, and mean diameter of particles on the physical stability and morphology of PLGA particles, and on their potential to be redispersed after storage. To observe potential changes in the extent of aggregation using confocal microscopy, PLGA nanospheres and microspheres were prepared containing a lipophilic, green fluorescent dye, Bodipy Fl C5 (?ex:495 nm; ?em:512 nm). In addition, scanning electron microscopy (SEM) was used to study changes in aggregation, coalescence, and solid-state surface morphology of the PLGA particles.
Materials
PLGA (50:50) with inherent viscosity of 0.69 dL/g (Lot No. 112-66-1) was obtained from Birmingham Polymers Inc (Birmingham, AL). Polyvinylalcohol (PVA), sodium chloride, sodium dihydrogen phosphate, and disodium hydrogen phosphate were obtained from Sigma Chemicals (St Louis, MO). BODIPY Fl C5 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, D-3834) was purchased from Molecular Probes (Eugene, OR).
Methods
Preparation of Nanospheres and Microspheres Containing Bodipy
Nanospheres containing Bodipy were prepared using the conventional emulsion-evaporation technique.20,21 Initially a 500-?g/mL solution of Bodipy was prepared in methanol. PLGA (90 mg) was dissolved in 3 mL of dichloromethane and 0.1 mL of Bodipy (50 ?g) in methanol was added. This solution was then emulsified for 60 seconds with 25 mL of 1.5% wt/vol PVA solution using a microtip probe sonicator at 36 W (Misonix Sonicator 3000 with microtip probe, Misonix Inc, Farmingdale, NY). Two sizes of nanospheres were prepared by emulsifying the 2 phases for 60 seconds at 36 or 10 W using a Misonix sonicator, while emulsification for 60 seconds using a Sorvall Omni-Mixer (Sorvall Instruments, Norwalk, CT) at 3 and 1 W formed larger microspheres. The dichloromethane was evaporated by stirring overnight, and the suspension ultracentrifuged at 140 000g for 25 minutes at 4?C (34 500 rpm using 50.2 Ti rotor). The remaining pellet was resuspended in water by sonicating for 30 seconds and washed twice with water. The washings were centrifuged at 130 000g (33 000 rpm) for 20 minutes at 4?C, and the pellet was resuspended by sonication and maintained at -80?C for 2 hours before being lyophilized for 36 hours. The resulting nanospheres were then stored in a dessicator at 4?C until used.
Characterization of Nanospheres and Microspheres
Determination of Particle Size and Size Distribution Using Dynamic Light Scattering
Nanospheres or microspheres (0.5 mg) were dispersed in 3 mL of water by sonication and added then to a 3-mL cuvette. The mean particle size ? SD, and zeta potential of each batch of particles were determined using the Zeta Plus dynamic light scattering particle size analyzer (Brookhaven Instrument Corp, Holtsville, NY).
Effect of Storage Temperature on the Aggregation of PLGA Particles of Varying Size
The Effect of Particle Size on the Changes in Morphology of PLGA Nanospheres and Microspheres Stored at 37?C
To establish if there was an effect of particle size on the morphology of PLGA particles containing Bodipy when stored at 37?C, nanospheres and microspheres were prepared with the following approximate diameters: 250 nm, 350 nm, 1 ?m, and 2 ?m. Samples of the PLGA-Bodipy particles were incubated at 37?C in 20-mL glass scintillation vials. After 6 days, 0.5 mg of these particles was suspended in 3 mL of water and visualized using a Zeiss confocal microscope (LSM 410 Confocal Laser Scanning Microscope [Goettinger, Germany]) with 1 filter at 488 nm and another cut-off filter at 515 nm. The duration of storage, 6 days, was selected based on previous time-dependent aggregation studies on particles in the same size range.
Effect of Storage Temperature on Nanosphere Aggregation
To study the effect of temperature on the extent of aggregation during storage, the smallest Bodipy-PLGA nanospheres (266 nm, 5 mg) contained in scintillation vials were incubated at 4?C, 25?C, 37?C, and 50?C for 6 days. The extent of aggregation of these nanospheres was monitored after 6 days using both confocal microscopy and SEM. In addition, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) thermograms of the Bodipy-PLGA particles at 0 and after 6 days were obtained using a Shimadzu DSC-50 and TGA-50 (Shimadzu Corporation, Kyoto, Japan) and compared with control pure PLGA polymer. The DSC and TGA thermograms were obtained after sealing 1 mg of particles, which had been stored at each temperature, in aluminum pans and heating at a rate of 10?C/min to 350?C in an atmosphere of nitrogen (20 mL/min).
Effect of PVA Concentration and Residual Dichloromethane on the Extent of Aggregation
To investigate if PVA influenced the extent of aggregation, Bodipy-PLGA nanospheres (300 nm) were prepared using the previously described protocol, except that the organic phase was emulsified with aqueous PVA solutions of 3 concentrations (0.75%, 1.5%, and 2.5% wt/vol). Samples (5 mg) from these 3 batches of nanospheres were stored in scintillation vials at 4?C, 25?C, 37?C, and 50?C for 6 days. After 6 days, the extent of aggregation and morphology of the nanospheres was observed using confocal microscopy and SEM, and their DSC and TGA thermograms were recorded.
To test if the aggregation of nanospheres may have been due to the residual dichloromethane within the particles, the particles were prepared according to the above protocol, except the suspension of nanospheres was placed in a rotary evaporator for 2 hours at 27?C after stirring overnight. The nanospheres were then washed to remove PVA and lyophilized as described previously. After lyophilization, samples (5 mg) of these nanospheres (266 nm), prepared using an additional rotavap step, were stored in scintillation vials at 37?C for 0, 1, and 2 days. To evaluate whether changes had occurred, nanospheres were then resuspended and observed using confocal microscopy, and the DSC and TGA thermograms were obtained. Nanospheres that had not been subjected to the additional evaporation step were used as control.
Results and Discussion
Preparation and Characterization of Various-Sized BODIPY-PLGA Particles and the Effect of Storing the Particles at 37?C on Aggregation
The mean diameter and SD of the batches of lyophilized Bodipy-PLGA particles (Figure 1A) were 266.9 ? 2.8, 351.6 ? 1.8, 988.8 ? 14.1, and 1865.9 ? 67.0 nm with corresponding Bodipy content of 0.026%, 0.027%, 0.028%, and 0.030% wt/wt, respectively. The particles were formulated using the standard emulsion-evaporation method and then lyophilized. After lyophilization, all particles were spherical with smooth surfaces. SEM photomicrographs of Bodipy-PLGA control (prior to storage) particles, independent of size, illustrated that all nanospheres and microspheres were discrete entities and compactly arranged after lyophilization (Figure 2). Of importance, additional SEM studies indicated that lyophilization caused the PLGA particles to align in intimate contact as fibers (Figure 3). This finding indicates that lyophilization did not cause aggregation of only the nanospheres. In addition, the a