Controlled release parenteral nanospheres and microspheres made from biodegradable polymers such as poly (lactide-co-glycolide) (PLGA) have been widely investigated for a variety of therapeutic agents. PLGA polymers have an excellent record of biocompatibility, biodegradability and non-immunogenicity [1
]. PLGA polymers have also been used in the preparation of controlled release delivery systems, such as Lupron Depot®
(TAP Pharmaceutical Product Inc.), Zoladex™ (Zeneca), Decapeptyl™ (Ipsen Biotech), and Prostap SR™ (Lederle), all of which are licensed for intramuscular use in humans in both Europe and the U.S.A.
Functionalized magnetic nanospheres and microspheres (MMS) are usually formulated by encapsulation of magnetic nanoparticles (e.g., magnetite) into the biodegradable polymeric matrix and are being used evermore for biomedical applications including drug delivery, diagnostic magnetic resonance imaging (MRI), magnetic cell separation, tissue repair, hyperthermia and magnetofection [4
]. Superparamagnetic iron oxide contrast agents have been approved by FDA for MRI. Commercial iron oxide nanoparticles of maghemite (Endorem®
) are used as contrast agents in MRI for the diagnosis and exact location determination of brain [9
] and cardiac infarcts [11
], and for the detection of liver lesions and tumors [12
], where the magnetic nanoparticles tend to accumulate at higher levels due to the difference in tissue composition and/or endocytotic uptake processes. In a clinical study, it was concluded that intracranial thermotherapy using magnetic nanoparticles can be safely applied on glioblastoma multiforme patients [13
]. The toxicity of magnetite encapsulated in PLGA has not been tested, although PLA microspheres containing 10% magnetite were tested in a rat model for treatment of intraspinal glioblastoma by delivering local radiation. After injection of 0.5 mg of microspheres through an intrathecal catheter, all 12 rats survived for 15 months with no apparent side effects or weight loss [14
]. Due to their magnetic component, MMS can be locally targeted using externally applied magnetic fields. A promising approach, for example, is the intravascular injection of magnetic particles (ferrofluids) bound to anticancer agents that are then concentrated in the desired area (e.g., a tumor) by an external magnetic field [15
]. It might thus be possible to overcome the systemic side effects of many chemotherapeutic agents.
We sought to develop biodegradable sustained release PLGA MMS which could be administered by intravascular injection and which are attracted by external magnetic fields to a specific site [6
]. For the desired mode of administration, the size of the MMS must be optimized. The MMS have to be smaller than red blood cells and must be delivered through blood vessels at concentrations which do not embolize (clog) the capillaries. Since the diameter of the smallest blood vessels, the capillaries, is typically 7–8 μm [16
], a particle size under 2 μm enables intravascular injection and is also desirable for intramuscular and subcutaneous administration, minimizing possible irritant reactions [17
]. On the non-physiological side, particle size also influences both the loading efficiency of the magnetic materials that turn microspheres into MMS and of the therapeutic agents. This is especially important if the loading efficiencies have to be maximized. In general, the larger the particle size, the higher the encapsulation efficiency for drugs and magnetic nanoparticles, as can be explained by the fact that the size increase is related to a relative decrease of the surface area, thus reducing the possibility of drug loss by diffusion during the fabrication procedure [17
]. In addition, the magnetic forces required to direct and localize the MMS will increase substantially with decreasing particle size, requiring that the microspheres be large enough to allow the use of reasonably sized magnets [18
Our work focuses on the effect of process and formulation variables in the preparation of magnetic PLGA microspheres in the specific size range of 1 to 2 μm. A couple of papers describe the use of large PLGA MMS of 20–50 μm [19
], while Lee et al. prepared magnetic PLGA nanoparticles (90–180 nm) by an emulsification-diffusion method after stabilizing iron oxide nanoparticles by sodium oleate and sodium dodecylbenzenesulfonate [21
]. Most recently, Liu et al. have successfully fabricated microspheres containing 45 weight% of oleic acid-coated magnetite using a polymer mixture of PLGA and a diblock copolymer of poly(lactic acid) and polyethylene glycol (PLA-PEG). They used a single emulsion method and loaded the magnetite into the oil phase [22
]. To our knowledge, there are no other studies looking at small PLGA MMS of 1–2 μm. In the field of non-magnetic microspheres, however, Muramatsu et al. have prepared uniform PLGA microspheres by forcing the polymer dissolved in methylene chloride through a glass membrane of homogeneous pore size of about 1120 nm [23
]. Two other studies examined the preparation conditions of PLA, PLGA and PLA-PEG-PLA particles incorporating DNA, in the size range of 200–700 nm [24
] and 1–2 μm [25
]. Still another method of preparing PLGA microspheres is acoustic excitation, resulting in over 95% of the spheres having a diameter of 1.0–1.5 μm [26
]. But none of these preparations involved the incorporation of magnetic materials.
In the present investigation, both non-magnetic and magnetic microspheres were prepared by a W/O/W double emulsion and solvent evaporation method. Effects of homogenization conditions (speed and time), inner water phase (volume and protein concentration), oil phase (volume, polymer concentration in oil phase and solvent composition), outer water phase (stabilizer concentration) and evaporation speed on particle size were systematically investigated. The size optimized method found during the preparation of non-magnetic microspheres was then applied to the making of MMS by loading water dispersible magnetite coated by polyethylene glycole/polyacrylic acid (PEG/PAA) into the inner water phase.