The Effective Solubilization of Hydrophobic Drugs Using Epigallocatechin Gallate or Tannic Acid-Based Formulations.

Hydrotropic solubilization of hydrophobic drugs requires supramolar amounts of hydrotropes with potential toxicity issues. We investigated the use of epigallocatechin gallate (EGCG) and tannic acid at millimolar concentrations, as hydrotrope-like solubilizing agents. Paclitaxel, docetaxel, amphotherecin B, curcumin, or rapamycin were dried down with EGCG or tannic acid from ethanol and then redissolved in aqueous media. Following centrifugation and filtration, the drug solubility was measured using HPLC. The uptake of docetaxel into cells from EGCG-based solutions was measured using radiolabeled drugs. Both EGCG and tannic acid effectively increased the aqueous solubility of all drugs from low levels (µg/mL) to high levels (mg/mL) in a concentration-dependent fashion at millimolar concentrations. Solutions were generally stable at room temperature over 24 h. Compared with micellar formulations, EGCG-based solutions of docetaxel demonstrated markedly improved drug uptake or transport levels in all cell lines. The use of these additives may provide improved formulation of various hydrophobic drugs using oral, parenteral, localized, or device-associated delivery systems.

Same-single-cell analysis using the microfluidic biochip to reveal drug accumulation enhancement by an amphiphilic diblock copolymer drug formulation.

Multidrug resistance (MDR) is one of the major obstacles in drug delivery, and it is usually responsible for unsuccessful cancer treatment. MDR may be overcome by using MDR inhibitors. Among different classes of these inhibitors that block drug efflux mediated by permeability-glycoprotein (P-gp), less toxic amphiphilic diblock copolymers composed of methoxypolyethyleneglycol-block-polycaprolactone (MePEG-b-PCL) have been studied extensively. The purpose of this work is to evaluate how these copolymer molecules can reduce the efflux, thereby enhancing the accumulation of P-gp substrates (e.g., daunorubicin or DNR) in MDR cells. Using conventional methods, it was found that the low-molecular-weight diblock copolymer, MePEG17-b-PCL5 (PCL5), enhanced drug accumulation in MDCKII-MDR1 cells, but the high-molecular-weight version, MePEG114-b-PCL200 (PCL200), did not. However, when PCL200 was mixed with PCL5 (and DNR) in order to encapsulate them to facilitate drug delivery, there was no drug enhancement effect attributable to PCL5, and the reason for this negative result was unclear. Since drug accumulation measured on different cell batches originated from single cells, we employed the same-single-cell analysis in the accumulation mode (SASCA-A) to find out the reason. A microfluidic biochip was used to select single MDR cells, and the accumulation of DNR was fluorescently measured in real time on these cells in the absence and presence of PCL5. The SASCA-A method allowed us to obtain drug accumulation information faster in comparison to conventional assays. The SASCA-A results, and subsequent curve-fitting analysis of the data, have confirmed that when PCL5 was encapsulated in PCL200 nanoparticles as soon as they were synthesized, the ability of PCL5 to enhance DNR accumulation was retained, thus suggesting PCL200 as a promising delivery system for encapsulating P-gp inhibitors, such as PCL5.

Mixed molecular weight copolymer nanoparticles for the treatment of drug-resistant tumors: formulation development and cytotoxicity.

Nanoparticles composed of both high- and low-molecular-weight methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL) diblock copolymers (termed "mixed molecular weight nanoparticles") were investigated for the encapsulation and delivery of the taxane drugs paclitaxel (PTX) and docetaxel (DTX). These nanoparticles were prepared using nanoprecipitation and emulsion methods. These 80 nm nanoparticles were prepared with high yields, efficiently solubilized PTX and DTX up to 500 and 1300 µg/mL, respectively, and demonstrated controlled release of these drugs over 14 days. The taxane-sensitive (MDCKII) and taxane-resistant [P-glycoprotein (P-gp) overexpressing] MDCKII-MDR cell lines were used to establish the cytotoxic profiles of these nanoparticles. Because of the coencapsulation of the previously demonstrated P-gp inhibitor, a low-molecular-weight MePEG-b-PCL copolymer (MePEG17 -b-PCL5 ), these drug-loaded mixed molecular weight nanoparticles dramatically reduced the viability of P-gp overexpressing MDCKII-MDR cells and restored sensitivity to taxane drugs in these cells.

Fusidic acid and rifampicin co-loaded PLGA nanofibers for the prevention of orthopedic implant associated infections.

Implant-associated infections following invasive orthopedic surgery are a major clinical problem, and are one of the primary causes of joint failure following total joint arthroplasty. Current strategies using perioperative antibiotics have been met with little clinical success and have resulted in various systemic toxicities and the promotion of antibiotic resistant microorganisms. Here we report the development of a biodegradable localized delivery system using poly(D,L-lactic acid-co-glycolic acid) (PLGA) for the combinatorial release of fusidic acid (FA) (or its sodium salt; SF) and rifampicin (RIF) using electrospinning. The drug-loaded formulations showed good antibiotic encapsulation (~75%-100%), and a biphasic drug release profile. All dual-loaded formulations showed direct antimicrobial activity in vitro against Staphylococcus epidermidis, and two strains of methicillin-resistant Staphylococcus aureus (MRSA). Furthermore, lead formulations containing 10% (w/w) FA/SF and 5% (w/w) RIF were able to prevent the adherence of MRSA to a titanium implant in an in vivo rodent model of subcutaneous implant-associated infection.

The combined use of paclitaxel-loaded nanoparticles with a low-molecular-weight copolymer inhibitor of P-glycoprotein to overcome drug resistance.

Two types of nanoparticles were prepared using the diblock copolymer methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL), with either a short PCL block length, which forms micelles, or with a longer PCL block length, which forms kinetically "frozen core" structures termed nanospheres. Paclitaxel (PTX)-loaded micelles and nanospheres were evaluated for their cytotoxicity, cellular polymer uptake, and drug accumulation in drug-sensitive (Madin-Darby Canine Kidney [MDCK]II) and multidrug-resistant (MDR) P-glycoprotein (P-gp)-overexpressing (MDCKII-MDR1) cell lines. Both types of PTX-loaded nanoparticles were equally effective at inhibiting proliferation of MDCKII cells, but PTX-loaded micelles were more cytotoxic than nanospheres in MDCKII-MDR1 cells. The intracellular accumulation of both PTX and the diblock copolymers were similar for both nanoparticles, suggesting that the difference in cytotoxicity might be due to the different drug-release profiles. Furthermore, the cytotoxicity of these PTX-loaded nanoparticles was enhanced when these systems were subsequently or concurrently combined with a low-molecular-weight MePEG-b-PCL diblock copolymer, which we have previously demonstrated to be an effective P-gp inhibitor. These results suggest that the dual functionality of MePEG-b-PCL might be useful in delivering drug intracellularly and in modulating P-gp in order to optimize the cytotoxicity of PTX in multidrug-resistant cells.

CONTROLLED DELIVERY OF ANTIANGIOGENIC DRUG TO HUMAN EYE TISSUE USING A MEMS DEVICE.

We demonstrate an implantable MEMS drug delivery device to conduct controlled and on-demand, ex vivo drug transport to human eye tissue. Remotely operated drug delivery to human post-mortem eyes was performed via a MEMS device. The developed curved packaging cover conforms to the eyeball thereby preventing the eye tissue from contacting the actuating membrane. By pulsed operation of the device, using an externally applied magnetic field, the drug released from the device accumulates in a cavity adjacent to the tissue. As such, docetaxel (DTX), an antiangiogenic drug, diffuses through the eye tissue, from sclera and choroid to retina. DTX uptake by sclera and choroid were measured to be 1.93±0.66 and 7.24±0.37 µg/g tissue, respectively, after two hours in pulsed operation mode (10 s on/off cycles) at 23°C. During this period, a total amount of 192 ng DTX diffused into the exposed tissue. This MEMS device shows great potential for the treatment of ocular posterior segment diseases such as diabetic retinopathy by introducing a novel way of drug administration to the eye.

Phase separation behavior of fusidic acid and rifampicin in PLGA microspheres.

The purpose of this study was to characterize the phase separation behavior of fusidic acid (FA) and rifampicin (RIF) in poly(d,l-lactic acid-co-glycolic acid) (PLGA) using a model microsphere formulation. To accomplish this, microspheres containing 20% FA with 0%, 5%, 10%, 20%, and 30% RIF and 20% RIF with 30%, 20% 10%, 5%, and 0% FA were prepared by solvent evaporation. Drug-polymer and drug-drug compatibility and miscibility were characterized using laser confocal microscopy, Raman spectroscopy, XRPD, DSC, and real-time video recordings of single-microsphere formation. The encapsulation of FA and RIF alone, or in combination, results in a liquid-liquid phase separation of solvent-and-drug-rich microdomains that are excluded from the polymer bulk during microsphere hardening, resulting in amorphous spherical drug-rich domains within the polymer bulk and on the microsphere surface. FA and RIF phase separate from PLGA at relative droplet volumes of 0.311 ± 0.014 and 0.194 ± 0.000, respectively, predictive of the incompatibility of each drug and PLGA. When coloaded, FA and RIF phase separate in a single event at the relative droplet volume 0.251 ± 0.002, intermediate between each of the monoloaded formulations and dependent on the relative contribution of FA or RIF. The release of FA and RIF from phase-separated microspheres was characterized exclusively by a burst release and was dependent on the phase exclusion of surface drug-rich domains. Phase separation results in coalescence of drug-rich microdroplets and polymer phase exclusion, and it is dependent on the compatibility between FA and RIF and PLGA. FA and RIF are mutually miscible in all proportions as an amorphous glass, and they phase separate from the polymer as such. These drug-rich domains were excluded to the surface of the microspheres, and subsequent release of both drugs from the microspheres was rapid and reflected this surface location.

The solid-state characterization of fusidic acid.

PURPOSE: The aim of this work was to characterize the solid-state properties of fusidic acid (FA). METHODS: Solid forms of FA were prepared by solvent-mediated polymorphic transformation of commercial FA (Form III) in acetonitrile (ACN), and methanol:H(2)O (50:50), or generated by solvent recrystallization from dichloromethane (DCM). Polymorphs were characterized using, X-ray diffraction (XRD), differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), polarizing hot stage microscopy (HSM), and intrinsic dissolution rate (IDR). RESULTS: Slurrying commercial FA (Form III) in methanol:H(2)O (50:50), yielded a metastable form (Form IV). This metastable form converts to Form I or back to Form III in ACN and H(2)O, respectively, and Form II upon recrystallization from DCM. IDR of Form IV was 0.092 mg/min/cm(2), and was statistically different (p<0.05) from the IDR of Forms I, II, and III, with IDR of 0.053, 0.043, and 0.045mg/min/cm(2), respectively. The amorphous FA had an IDR of 0.125 mg/min/cm(2), and was significantly higher (p<0.05) than any other solid form. There were no statistical differences in the IDR of Form I, II, or III. CONCLUSIONS: This work provides evidence for the existence of two previously unreported polymorphic forms of FA (Forms II and IV) and an amorphate.

Copolymer micelles and nanospheres with different in vitro stability demonstrate similar paclitaxel pharmacokinetics.

Paclitaxel loaded amphiphilic block copolymer nanoparticles have been demonstrated to enhance the aqueous solubility and improve the toxicity profile as compared to the commercially available product Taxol; however, in many cases long circulation of the drug is not achieved due to rapid partitioning of the drug from the carrier and/or carrier instability upon injection. In this work we investigated the effect of increasing the hydrophobic block length of methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (MePEG-b-PCL) copolymers on the physicochemical properties and in vitro stability of the formed nanoparticles as well as the pharmacokinetics and biodistribution of both the copolymer and solubilized drug. We hypothesized that copolymers composed of high molecular weight hydrophobic blocks (MePEG114-b-PCL104) that form nanoparticles with a kinetically "frozen core" (which we term nanospheres) would better retain their PTX payload as compared to micelles composed of shorter hydrophobic blocks (MePEG114-b-PCL19), thus leading to prolonged drug circulation. Nanospheres solubilized PTX more efficiently, released the drug in a more sustained fashion and were characterized by enhanced stability and drug retention in the presence of plasma proteins as compared to micelles. Using radiolabeled copolymers and PTX, it was found that, upon injection, MePEG114-b-PCL104 circulated for longer than MePEG114-b-PCL19; however, the drug was rapidly eliminated from the blood regardless of the formulation. These results suggest that, despite formulation in more stable nanospheres, PTX was still rapidly extracted from these nanoparticles.

The use of nanocrystalline cellulose for the binding and controlled release of drugs.

The objective of this work was to investigate the use of nanocrystalline cellulose (NCC) as a drug delivery excipient. NCC crystallites, prepared by an acid hydrolysis method, were shown to have nanoscopic dimensions and exhibit a high degree of crystallinity. These crystallites bound significant quantities of the water soluble, ionizable drugs tetratcycline and doxorubicin, which were released rapidly over a 1-day period. Cetyl trimethylammonium bromide (CTAB) was bound to the surface of NCC and increased the zeta potential in a concentration-dependent manner from -55 to 0 mV. NCC crystallites with CTAB-modified surfaces bound significant quantities of the hydrophobic anticancer drugs docetaxel, paclitaxel, and etoposide. These drugs were released in a controlled manner over a 2-day period. The NCC-CTAB complexes were found to bind to KU-7 cells, and evidence of cellular uptake was observed.

Synthesis and characterization of carboxylic acid conjugated, hydrophobically derivatized, hyperbranched polyglycerols as nanoparticulate drug carriers for cisplatin.

Hyperbranched polyglycerols (HPGs) with hydrophobic cores and derivatized with methoxy poly(ethylene glycol) were synthesized and further functionalized with carboxylate groups to bind and deliver cisplatin. Low and high levels of carboxylate were conjugated to HPGs (HPG-C(8/10)-MePEG(6.5)-COOH(113) and HPG-C(8/10)-MePEG(6.5)-COOH(348)) and their structures were confirmed through NMR and FTIR spectroscopy and potentiometric titration. The hydrodynamic diameter of the HPGs ranged from 5-10 nm and the addition of COOH groups decreased the zeta potential of the polymers. HPG-C(8/10)-MePEG(6.5)-COOH(113) bound up to 10% w/w cisplatin, whereas HPG-C(8/10)-MePEG(6.5)-COOH(348) bound up to 20% w/w drug with 100% efficiency. Drug was released from HPG-C(8/10)-MePEG(6.5)-COOH(113) over 7 days at the same rate, regardless of the pH. Cisplatin release from HPG-C(8/10)-MePEG(6.5)-COOH(348) was significantly slower than HPG-C(8/10)-MePEG(6.5)-COOH(113) at pH 6 and 7.4, but similar at pH 4.5. Release of cisplatin into artificial urine was considerably faster than into buffer. Carboxylated HPGs demonstrated good biocompatibility, and drug-loaded HPGs effectively inhibited proliferation of KU-7-luc bladder cancer cells.

In vitro human plasma distribution of nanoparticulate paclitaxel is dependent on the physicochemical properties of poly(ethylene glycol)-block-poly(caprolactone) nanoparticles.

In this study, we synthesized and characterized two methoxy poly(ethylene glycol)-block-poly(caprolactone) (MePEG-b-PCL) amphiphilic diblock copolymers, both based on MePEG with a molecular weight of 5000 g/mol (114 repeat units) and PCL block lengths of either 19 or 104 repeat units. Nanoparticles were formed from these copolymers by a nanoprecipitation and dialysis technique. The MePEG(114)-b-PCL(19) copolymer was water soluble and formed micelles that had a hydrodynamic diameter of 40 nm at all copolymer concentrations tested, and displayed a relatively low core microviscosity. The practically water insoluble MePEG(114)-b-PCL(104) copolymer formed nanoparticles with a larger hydrodynamic diameter, which was dependent on copolymer concentration, and possessed a higher core microviscosity than the MePEG(114)-b-PCL(19) micelles, characteristic of nanospheres. The micelles solubilized a maximum of 1.6% w/w of the hydrophobic anticancer agent, paclitaxel (PTX), and released 92% of their drug payload over 7 days, as compared to the nanospheres, which solubilized a maximum of 3% w/w of PTX and released 60% over the same period of time. Both types of nanoparticles were found to be hemocompatible, causing only minimal hemolysis and no changes in plasma coagulation times as compared to control. Upon in vitro incubation in human plasma, PTX solubilized by micelles had a plasma distribution similar to free drug. The majority of PTX was associated with the lipoprotein deficient plasma (LPDP) fraction, which primarily consists of albumin and alpha-1-acid glycoprotein. In contrast, nanospheres were capable of retaining more of the encapsulated drug with significantly less PTX partitioning into the LPDP fraction.

Solubilization of hydrophobic drugs by methoxy poly(ethylene glycol)-block-polycaprolactone diblock copolymer micelles: theoretical and experimental data and correlations.

The solubilization of five model hydrophobic drugs by a series of micelle-forming, water-soluble methoxy poly(ethylene glycol)-block-polycaprolactone diblock copolymers (MePEG-b-PCL) with varying methoxy poly(ethylene glycol) (MePEG) and polycaprolactone (PCL) block lengths was investigated. Variation of the feed weight ratio of MePEG to caprolactone resulted in the synthesis of copolymers with predictable block lengths. The micelle diameter and pyrene partition coefficient (Kv) were directly related to the PCL block length whereas the critical micelle concentrations (CMC) were inversely related to the PCL block length. The aqueous solubilities of the model hydrophobic drugs, indomethacin, curcumin, plumbagin, paclitaxel, and etoposide were increased by encapsulation within the micelles. Drug solubilization was directly related to the compatibility between the solubilizate and PCL as determined by the Flory-Huggins interaction parameter (chisp). Furthermore, the concentration of solubilized drug was also directly related to the PCL block length.

The characterization of paclitaxel-loaded microspheres manufactured from blends of poly(lactic-co-glycolic acid) (PLGA) and low molecular weight diblock copolymers.

Paclitaxel-loaded biodegradable drug delivery systems manufactured from poly(lactic-co-glycolic acid) (PLGA) are known to release the drug at extremely slow rates. The objective of this study was to characterize paclitaxel-loaded microspheres composed of blends of PLGA with low molecular weight ampipathic diblock copolymers. The encapsulation and release of a series of poly(epsilon-caprolactone) (PCL)- or poly(D,L-lactic acid) (PDLLA)-co-methoxypolyethylene glycol (MePEG) diblock copolymers was measured using quantitative gel permeation chromatography. Polymeric miscibility was determined by glass transition temperature measurements using differential scanning calorimetry and paclitaxel release was measured using HPLC methods. The PCL- and PDLLA-based diblock copolymers encapsulated at high efficiency and were miscible in PLGA microspheres (30-120m microm size range). The burst phase of paclitaxel release was increased up to 20-fold by the inclusion of diblock copolymers in PLGA microspheres. Approximately 10% of the more hydrophobic PCL-based copolymers released from the microspheres in a short burst over 3 days followed by very slow release over the following 10 weeks. Only the PDLLA-based copolymer released from the PLGA microspheres in a controlled manner over 10 weeks. All microspheres containing PEG were found to have more hydrophilic surfaces (as measured by contact angle) with improved biocompatibility (reduced neutrophil activation) compared to PLGA only microspheres. These results indicate that low molecular weight polyester-based diblock copolymers may be effectively encapsulated in PLGA microspheres to increase paclitaxel release (probably through a micellization process) and improve biocompatibility.

A review of the formation and classification of amphiphilic block copolymer nanoparticulate structures: micelles, nanospheres, nanocapsules and polymersomes.

Amphiphilic block copolymers are able to form a range of different nanoparticulate structures. These include micelles, nanospheres, nanocapsules, and polymersomes. This review attempts to clarify some of the terminology used in the literature by providing an overview of the major features of each type of nanoparticle and the factors that influence the formation of particular nanoparticulate formulations.

Determining the critical micelle concentration of a novel lipid-lowering agent, disodium ascorbyl phytostanyl phosphate (FM-VP4), using a fluorescence depolarization procedure.

The objective of this study was to determine the critical micelle concentration (CMC) of a novel water-soluble plant sterol derivative (FM-VP4) using a fluorescence depolarization method. The CMC was determined by 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence depolarization. Test solutions of various concentrations of sodium dodecylsulphate (SDS) as a positive control or FM-VP4 in water were spiked with 2 microL of 4 mM DPH in tetrahydrofuran (THF) and left overnight to equilibrate in a dark chamber. Fluorescence of each solution was measured at room temperature using a Perseptive Biosystems Cytofluor Series 4000 multi-well plate reader. Fluorescence intensity increases as DPH is incorporated into the hydrophobic core of micelles. Thus, the CMC is the value at which an abrupt increase in intensity is observed. These points were observed at 8 mM and 0.014 mM for SDS and FM-VP4, respectively. Sodium dodecylsulphate was used as a positive control and supports the validity of our results, as the literature values of SDS are reported to be between 8-8.3 mM. The CMC of FM-VP4 is reported to be 0.014 mM.

Characterization of perivascular poly(lactic-co-glycolic acid) films containing paclitaxel.

The objectives of this study were to investigate the use of poly(lactic-co-glycolic acid) (PLGA) for the formulation of paclitaxel loaded films and to characterize these films for potential application as perivascular "wraps" to prevent restenosis. Films were manufactured from PLGA blended with either methoxypolyethylene glycol (MePEG) or a diblock copolymer composed of poly(D,L-lactic acid)-block-methoxypolyethylene glycol, PDLLA-MePEG (diblock) by solvent evaporation on teflon discs. Elasticity was determined by gravimetric stress/strain analysis. Thermal analysis was determined using differential scanning calorimetry (DSC). Changes in film composition and degradation in aqueous media were determined using gel permeation chromatography (GPC). Paclitaxel release from films was measured by incubation of the films in phosphate buffered saline (PBS) with drug analysis by HPLC methods. The addition of MePEG or diblock to PLGA caused a concentration dependent increase in the elasticity of films, due to plasticizing effects. DSC analysis showed that MePEG and diblock caused a concentration dependent decrease in the glass transition temperature (Tg) of PLGA indicating miscibility of the polymers. When placed in aqueous media, more than 75% of MePEG dissolved out of the PLGA films within 2 days, whereas diblock partitioned slowly and in a controlled manner out of the films. Paclitaxel release from PLGA/MePEG films was very slow with less than 5% of the encapsulated drug being released over 2 weeks. The addition of 30% diblock to paclitaxel loaded PLGA films caused a substantial increase (five- to eight-fold) in the release rate of paclitaxel. PLGA films containing 30% diblock and either 1% or 5% paclitaxel were partially or completely degraded following perivascular implantation in rats.

Synthesis and micellar characterization of short block length methoxy poly(ethylene glycol)-block-poly(caprolactone) diblock copolymers.

A series of short block length methoxy poly(ethylene glycol)-block-poly(caprolactone) diblock copolymers was synthesized and characterized in order to assess the potential of these copolymers as a micellar drug-delivery system. Varying the caprolactone:MePEG weight ratio in the reaction mixture allowed the synthesis of diblock copolymers with a MePEG molecular weight of 750 g/mol and PCL block lengths of 2, 5 or 10 repeat units. Phase diagrams of aqueous solutions of the copolymers were constructed which displayed characteristic cloud points and Krafft points. As the degree of polymerization of PCL increased, critical micelle concentration (CMC) values decreased from 6.97 x 10(-1) to 3.38 x 10(-3) g/l, partition equilibrium coefficients (Kv) increased from 1.09 x 10(4) to 22.2 x 10(4),and hydrodynamic diameters increased from 12.2 to 19.5 nm. The micelle morphology was determined to be spherical by transmission electron microscopy.