Glen S. Kwon

Polymeric micelles as new drug carriers

Abstract Advances in block copolymer syntheses have led to polymeric micelles that may serve as nanoscopic drug carriers. For drug delivery, micelles were prepared from biocompatible and biodegradable block copolymers. The polymeric micelles display functional groups on their surfaces for attachment of pilot molecules. Researchers are establishing chemical as well as physical routes of loading drugs into polymeric micelles. Notably, polymeric micelles solubilize hydrophobic drugs suffering from poor water solubility. Recent studies suggest that polymeric micelles may have solid-like cores. Thus, they may remain intact for long durations under sink conditions and may also slowly release drugs. Mechanistically, polymeric micelles may act as drug carriers by circumventing host defenses, circulating for prolonged periods and extravasating from the vascular system, preferentially delivering drug to solid tumors.

Block copolymer micelles for drug delivery : loading and release of doxorubicin

Abstract Micelles of poly(ethylene oxide)-block-poly(β-benzyl- l -aspartate) (PEO-PBLA) were loaded with doxorubicin (DOX) and were characterized in relation to their use as drug vehicles. First, an oil-in-water emulsion method was developed to load DOX in PEO-PBLA micelles. The level of DOX in PEO-PBLA micelles was 5–12% w/w. Whereas the mean diameter of unloaded, PEO-PBLA micelles was ca., 19 nm, the mean diameter of PEO-PBLA micelles loaded with DOX was ≈37 nm. Minimal chemical degradation of DOX occurred as a result of loading in PEO-PBLA micelles. In addition, DOX in PEO-PBLA micelles was less susceptible to chemical degradation than free DOX in aqueous solution. There was evidence for retention of DOX in PEO-PBLA micelles even after freeze-drying and reconstitution in water. Lastly, PEO-PBLA micelles served as drug depots, slowly releasing DOX (days), even in the presence of 10% w/v serum albumin. The results suggest a number of pharmaceutical advantages of PEO-PBLA micelles for the delivery of DOX.

Polymeric micelles for drug delivery

Polymeric micelles are nanoscopic core/shell structures formed by amphiphilic block copolymers. Both the inherent and modifiable properties of polymeric micelles make them particularly well suited for drug delivery purposes. An emphasis of this review has been placed on both the description and characterization techniques of the physical properties of polymeric micelles. Relevant properties discussed include micellar association, morphology, size and stability. These properties and characterization techniques are included to provide context for the known advantages and applications of polymeric micelles for drug delivery. The advantages and applications discussed include solubilization of poorly soluble molecules, sustained release and size advantages, and protection of encapsulated substances from degradation and metabolism. The three most widely studied block copolymer classes are characterized by their hydrophobic blocks, and are poly(propylene oxide), poly(L-amino acid)s and poly(ester)s. These three classes of block copolymers are reviewed with multiple examples of current research in which formulation techniques with polymeric micelles have been applied to some of the most challenging molecules in the pharmaceutical industry. The polymeric micelles used for drug delivery in these examples have shown the abilities to attenuate toxicities, enhance delivery to desired biological sites and improve the therapeutic efficacy of active pharmaceutical ingredients.

Physical Entrapment of Adriamycin in AB Block Copolymer Micelles

The entrapment of Adriamycin (ADR) in micelles composed of AB block copolymers (poly(ethylene oxide-co-β-benzyl L-aspartate) (PEO-PBLA)) was investigated. The loading process involved transfer of ADR and PEO-PBLA into an aqueous milieu from dimethyl-formamide (DMF) through a dialysis procedure. Evidence for the physical entrapment of ADR in the polymeric micelles was derived from fluorescence spectroscopy and gel permeation chromatography (GPC). The total fluorescence intensity of ADR was low, suggesting that the drug was self-associated in the micelles. In addition, quenching experiments, using a water-soluble quencher (iodide (I–)), showed that the fluorescence of ADR present in micellar solutions was largely unaffected by I–, whereas the fluorescence of free ADR was readily quenched. From Stern-Volmer plots, quenching constants (KSV) of 2.2 and 17 M−l were determined for ADR in micellar solutions and free ADR, respectively. As a result of the entrapment of ADR in the micelles, ADR binds only slightly serum albumin as evidenced by GPC. In contrast, ADR readily binds serum albumin in aqueous solutions. The findings suggest that ADR is stably entrapped in PEO-PBLA micelles. ADR entrapment in polymeric micelles is expected to affect markedly the pharmacokinetics of ADR.

Encapsulation of plasmid DNA in biodegradable poly(d,l-lactic-co-glycolic acid) microspheres as a novel approach for immunogene delivery

A plasmid DNA encoding bacterial beta-galactosidase gene was encapsulated in poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres. Plasmid DNA extracted from PLGA microspheres retained both structural and functional integrity as evidenced by its restriction endonuclease digestion pattern and its ability to transfect COS-1 cells in vitro. PLGA microspheres protected plasmid DNA from digestion by deoxyribonuclease I (DNase I) in vitro. The encapsulation efficiency of plasmid DNA and its release rate depended on the molecular mass of PLGA. Lastly, J-774A macrophages phagocytosed PLGA microspheres loaded with plasmid DNA. Co-encapsulated monophosphoryl lipid A increased the rate of phagocytosis. These results suggest that biodegradable PLGA microspheres can deliver intact and functional plasmid DNA at controlled rates. Thus, PLGA microspheres may be used to jointly deliver genes and other biologically active molecules, e.g., immunomodulators, to antigen presenting cells.

Biodistribution of Micelle-Forming Polymer–Drug Conjugates

Polymeric micelles have potential utility as drug carriers. To this end, polymeric micelles based on AB block copolymers of polyethylene oxide (PEG) and poly(aspartic acid) [p(Asp)] with covalently bound Adriamycin (ADR) were prepared. The micelle forming polymer–drug conjugates [PEO-p(Asp(ADR)] were radiolabeled and their biodistribution was investigated after intravenous injection in mice. Long circulation times in blood for some compositions of PEO-p[Asp(ADR)] conjugates were evident, which are usually atypical of colloidal drug carriers. This was attributed to the low interaction of the PEO corona region of the micelles with biocomponents (e.g., proteins, cells). Low uptake of the PEO-p(Asp(ADR)] conjugates in the liver and spleen was determined. The biodistribution of the PEO-p[Asp(ADR)] conjugates was apparently dependent on micelle stability; stable micelles could maintain circulation in blood, while unstable micelles readily formed free polymer chains which rapidly underwent renal excretion. Long circulation times in blood of PEO-p(Asp(ADR)] conjugates are thought to be prerequisite for enhanced uptake at target sites (e.g., tumors).

Analysis of poly(D, L-lactic-co-glycolic acid) nanosphere uptake by human dendritic cells and macrophages in vitro

AbstractPurpose. The purpose of this study was to demonstrate and characterize phagocytosis of poly(D,L-lactic-co-glycolic acid) (PLGA) nanospheres by human dendritic cells (DCs). Methods. Parallel cultures of DCs and macrophages (Mφ) were established from peripheral blood leukocytes using media supplemented with granulocyte-macrophage colony stimulator factor and interleukin-4 (for DC) or granulocyte-macrophage colony stimulator factor alone (for Mφ). PLGA nanospheres containing tetramethylrhodamine-labeled dextran with or without an adjuvant, monophosphoryl lipid A, were prepared using a water/oil/water solvent evaporation technique. Cells were incubated with the nanospheres for 24 h. Confocal laser scanning microscopy was used to determine the intracellular location of the nanospheres and flow cytometry to measure the fraction of phagocytic cells in the culture and the amount of uptake per cell. After phagocytosis, cells were stained for MHC class II molecules, CD14, CD80, and CD86 to identify the phagocytic population. Results. DCs phagocytosed PLGA nanospheres as efficiently as Mφ. Cell-surface marker expression conclusively established that the phagocytic cells were DC. Conclusions. DCs can take up PLGA nanospheres. Because DCs are the key professional antigen-presenting cells capable of stimulating naive T cells, our data suggest that PLGA nanospheres can be used as an efficient delivery system for vaccines designed to activate T cell-mediated immune responses.

Polymeric micelles for drug delivery : solubilization and haemolytic activity of amphotericin B

Polymeric micelles may serve as nanoscopic, long-circulating carriers of hydrophobic drugs. In this study, we have researched the solubilization of amphotericin B (AmB), an antifungal drug, by micelles of poly(ethylene oxide)-block-poly(beta benzyl-L-aspartate) (PEO-PBLA), the properties of the AmB-loaded PEO-PBLA micelles and the resultant haemolytic activity of AmB. AmB loading takes place during self assembly of PEO-PBLA micelles, and this occurs through a dialysis procedure as an alkaline aqueous solution replaces the selective solvent for the polymer and the drug. In this way, AmB reaches levels of 57 to 141 microg/ml, corresponding to a loading efficiency of 27-30% (loaded AmB/initial amount of AmB). The molar ratio of AmB to PEO-PBLA is 0.40 to 1.0. Pictures by transmission electron microscopy reveal spherical AmB-loaded PEO-PBLA micelles with a mean diameter of 25.8+/-4.2 nm. AmB-loaded PEO-PBLA micelles are nonhaemolytic at an AmB level of 10 microg/ml as assessed by release of haemoglobin, measured by UV-Vis spectroscopy. AmB as Fungizone, its standard formulation, completely lyses red blood cells at a level of 3.0 microg/ml in 30 min. In contrast, there is no haemolysis at 5.5 h for AmB-loaded PEO-PBLA micelles at 3.0 microg/ml of AmB, indicating the gradual release of AmB from PEO-PBLA micelles. PEO-PBLA itself is nonhaemolytic even at a level of 0.70 mg/ml. Most amphiphiles, e.g. sodium deoxycholate, present in Fungizone, are haemolytic. Finally, AmB-loaded PEO-PBLA micelles can be freeze-dried and easily reconstituted in water. Afterwards, AmB is present in the intact PEO-PBLA micelles and remains nonhaemolytic.

Ovalbumin peptide encapsulated in Poly(d,l lactic-co-glycolic acid) microspheres is capable of inducing a T helper type 1 immune response

An ovalbumin (OVA) peptide, consisting of residues 323-339, was incorporated into poly(d,l lactic-co-glycolic acid) (PLGA) microspheres and administered to mice. It was hypothesized that microencapsulation of the peptide in PLGA microspheres would avoid the need for traditional adjuvants and bias the immune response towards a type 1 T helper (Th1) response. An immunomodulator, monophosphoryl lipid A (MPLA), was incorporated into the microspheres to determine its efficacy in enhancing a Th1 response. The specificity of the immune response was determined using a T cell proliferation assay. The type of T helper response was determined by analysis of the cytokine secretion profiles of the proliferating T cells. Following s.c. immunization, the results revealed a T cell-specific immune response for the encapsulated OVA peptide both with and without MPLA. The cytokine profiles revealed high levels of IFN-gamma with very low levels of IL-4 and IL-10, suggesting a Th1 response. Furthermore, incorporation of MPLA in the peptide loaded PLGA microspheres resulted in an increase in the production of IFN-gamma. Hence, peptide-loaded PLGA microspheres are capable of eliciting a specific Th1 immune response, which may be further enhanced in the presence MPLA.

Induction of anti-idiotypic humoral and cellular immune responses by a murine monoclonal antibody recognizing the ovarian carcinoma antigen CA125 encapsulated in biodegradable microspheres

Abstract The use of biodegradable poly(dl-lactic-co-glycolic acid) microspheres as a cancer vaccine delivery system for induction of anti-idiotypic responses was investigated using a murine monoclonal antibody B43.13 that recognizes the human ovarian cancer antigen CA125. Immunization of mice with mAb B43.13 encapsulated in poly(dl-lactic-co-glycolic acid) microspheres resulted in enhanced humoral and cellular immune responses compared with mAb B43.13 alone or mAb B43.13 mixed with microspheres. The antibody responses could be further enhanced by the co-encapsulation of mAb B43.13 with monophosphoryl lipid A, a non-toxic adjuvant, in microspheres. Anti-idiotypic humoral responses were shown to result in Ab2 antibodies mimicking the nominal antigen CA125 and Ab3 antibodies recognizing CA125. Further, microsphere delivery of mAb B43.13 also resulted in induction of T cell responses involving T2 cells reactive with mAb B43.13 epitopes and T3 cells recognizing CA125. These results indicate that microsphere delivery of Ab1 can induce both humoral and cellular anti-idiotypic responses relevant to cancer antigens. This raises the possibility of the use of such formulations for anti-idiotypic induction immunotherapy for cancer.