An acid-cleavable linker stable at neutral pH that releases doxorubicin at lysosomal pH

A Kemp's triacid amide of doxorubicin (DOX), esterified at a second carboxyl, releases DOX at lysosomal pH at a reasonable rate but is extremely stable at neutral pH.     

Semitelechelic HPMA copolymers functionalized with triphenylphosphonium as drug carriers for membrane transduction and mitochondrial localization

Semitelechelic HPMA (N-(2-hydroxypropyl)methacrylamide) copolymers possessing a single terminal lipophilic triphenylphosphonium (TPP) cation and fluorescent labels were synthesized to determine how the attached cation affected cellular uptake and intracellular trafficking. In vitro mitochondrial uptake fluorescence quenching assays using isolated mouse liver mitochondria indicated that only lower molecular weight (<5 kDa) BODIPY FL-labeled TPP-semitelechelic HPMA copolymers exhibited significant organelle localization or uptake. In vitro cellular uptake and intracellular trafficking was evaluated using cultured human ovarian carcinoma cells. Cells incubated with all types of TPP copolymers used in the study appeared to internalize the polymer by endocytosis only, and all of the internalized copolymer was confined to the lysosomal compartment after 24 h. Endocytotic uptake of the TPP-HPMA copolymer conjugates was rapid, suggesting that they were internalized by adsorptive endocytosis, rather than fluid-phase pinocytosis. Low-molecular weight (<5 kDa) and high-molecular weight (>5 kDa) semitelechelic copolymers, microinjected into cultured cells indicated that the TPP moiety did not significantly localize the polymers to mitochondria.     

Synthesis and biopharmaceutical characterisation of new poly(hydroxyethylaspartamide) copolymers as drug carriers.

Four new poly(hydroxyethylaspartamide)-based copolymers bearing (a) poly(ethylene glycol) 2000, (b) poly(ethylene glycol) 5000, (c) poly(ethylene glycol) 2000 and hexadecylalkyl, (d) poly(ethylene glycol) 5000 and hexadecylalkyle, as pendant groups were synthesised. The copolymers were obtained by partial aminolysis of polysuccinimide with poly(ethylene glycol) and hexadecylalkyl amino derivatives followed by reaction with ethanolamine. Naked polyhydroxyaspartamide was obtained by polysuccinimide reaction with ethanolamine. The nuclear magnetic resonance, infrared, light scattering and elemental analysis allowed for the extensive physico-chemical characterisation of the carriers. The molecular mass of all the polymers was in the range of 27000-34000 Da, and the polydispersivity was in the range of 1.5-1.7. By intravenous injection to mice bearing a solid tumour, all the polymeric carriers displayed a bi-compartmental pharmacokinetic behaviour. Both the poly(ethylene glycol) and the hexadecylalkyle conjugation prolonged and enhanced the distribution phase of poly(hydroxyethylaspartamide). The poly(ethylene glycol) conjugation was found to promote the carrier elimination by kidney ultrafiltration and to prevent partially the accumulation in the spleen and in the liver. The poly(ethylene glycol)/hexadecylalkyle conjugates localised preferentially in the liver were over 30% of the dose/g of tissue was determined after 144 h from administration. In the tumour all the polymers displayed a relevant accumulation that significantly increased throughout the time to reach high concentrations after 24 h. In particular, the poly(ethylene glycol)/hexadecylalkyle conjugates achieved a concentration of 15-25% of the dose/g of tissue after 24 h from administration that was maintained up to 144 h.     

Biodegradable amphiphilic copolymers based on poly(epsilon-caprolactone)-graft chondroitin sulfate as drug carriers

The goal of this study was to develop a new type of core-shell micelles based on biocompatible and biodegradable amphiphilic copolymers, named PCL-CS, using chondroitin sulfate (CS) as a hydrophilic segment and poly(epsilon-caprolactone) (PCL) as a hydrophobic segment. The copolymers, prepared from the various compositions between CS and PCL, were characterized by Fourier transform infrared spectrometer, proton nuclear magnetic resonance spectrometer, and differential scanning calorimeter. The PCL-CS copolymers could be assembled into micelles using a simple emulsion. With the fluorescent probe technique, the critical micelle concentrations were obtained in the range of 1.26 x 10(-3)-8.86 x 10(-3) mg/mL. The spherical images of micelles were visualized in the presence of polyvinyl alcohol (PVA) with the use of the transmission electron microscope. The particle sizes of micelles were all smaller than 300 nm, neither aggregate nor change in hydrodynamic sizes after 15 days staying in solutions containing salts or PVA by dynamic light scattering. The intracellular uptake of KB cells incubated with PCL-CS micelles was evidenced by confocal laser scanning microscope upon loading fluorescein isothiocyanate labeled bovine serum albumin as a probe.     

Liposomes as drug carriers.

The use of liposomes as drug carrying vehicles are summarized. Introduction of smaller doses, increased permeability, reduced toxicological and allergic effects and altered immunological responses are the advantages of the method. Experimental techniques for the preparation of drug containing liposomes and for their deliveries are surveyed. Proposed mechanisms for the incorporation of liposomes in cells are discussed. Examples for $\text{in vivo}$ applications and for functional liposomes capable of “homing-in” to given targets are presented.     

Long-living liposomes as potential drug carriers

Neutral, unilamellar liposomes (vesicles) composed of a dialkyl analog of phosphatidylcholine and cholesterol, and containing ¹⁴C-maltose as entrapped marker, were administered intravenously to mice. After one and two days, radioactivity in blood and liver remained 3–4 times higher than after administration of liposomes of egg (diester) phosphatidylcholine and cholesterol. It appears that the vesicles were taken into liver cells by endocytosis, and that phospholipases are involved in the capture as well as in the breakdown of conventional liposomes. Liposomes that are semi-resistant to catabolic enzymes may become useful in the manipulation of drug delivery.     

Photocrosslinkable biodegradable responsive hydrogels as drug delivery systems

Recently, controlled release from biocompatible materials has received much attention for biomedical applications. Due to their biocompatibility and biodegradability, glucopyranosides such as dextran appear as promising polymeric materials if one is able to regulate their rheological properties and the encapsulation/release efficiency. In this work graft polymer hydrogels from dextran and N-isopropylacrylamide (NIPAAm) were prepared and characterized.Dextran molecules were modified with 2-isocyanatoethylmethacrylate (IEMA) in order to obtain a polymer with carbon double bonds. Urethane linkages resulted from the reaction between hydroxyl groups (OH) of the dextran and isocyanate groups (NCO) of the IEMA. The obtained polymer was then crosslinked by UV irradiation in the presence of the photoinitiating agent Irgacure 2959 by CIBA^®. The drug Ondansetron^® was entrapped in the final system and its release profile was determined at 25 and 37 °C.The characterization of the materials was accomplished by: ATR-FTIR (Attenuated Total Reflectance-Fourier Transform Infrared) spectroscopy, elemental analysis, lower critical solution temperature (LCST) determination, swelling behaviour evaluation, determination of surface energy by contact angle measurement and drug delivery profile studies.     

Liposomes as drug carriers in leishmaniasis and malaria

Experimental studies suggest that liposomes could substantially improve the performance of anti-leishmanial drugs in the chemotherapy of visceral leishmaniasis. In this article, Carl Alving discusses the potential for overcoming resistance to antimonial drugs; for 'buffering' the toxicity of drugs, and for drug delivery under conditions where hospitalization is impossible or inconvenient. Liposomes can also be used experimentally to reduce the toxicity and increase the efficacy of parenterally-administered primaquine in the treatment of sporozoite-induced murine malaria.     

Synthesis of amphiphilic star block copolymers and their evaluation as transdermal carriers

Amphiphilic star polymers offer substantial promise for a range of drug delivery applications owing to their ability to encapsulate guest molecules. One appealing but underexplored application is transdermal drug delivery using star block copolymer reverse micelles as an alternative to the more common oral and intravenous routes. We prepared 6- and 12-arm amphiphilic star copolymers via atom transfer radical polymerization (ATRP) of sequential blocks of polar oligo (ethylene glycol)methacrylate and nonpolar lauryl methacrylate from brominated dendritic macroinitiators based on 2,2-bis(hydroxymethyl) propionic acid. These star block copolymers demonstrate the ability to encapsulate polar dyes such as rhodamine B and FITC-BSA in nonpolar media via UV/vis spectroscopic studies and exhibit substantially improved encapsulation efficiencies, relative to self-assembled "1-arm" linear block copolymer analogs. Furthermore, their transdermal carrier capabilities were demonstrated in multiple dye diffusion studies using porcine skin, verifying penetration of the carriers into the stratum corneum.     

Polyamidoamine dendrimers with different surface charge as carriers in anticancer drug delivery

Second-generation (G2) polyamidoamine (PAMAM) dendrimers are branched polymers containing 16 surface primary amine groups. Due to their structural properties, these polymers can be used as universal carriers in various drug delivery systems. Amine-terminated PAMAM dendrimers are characterized by a high positive surface charge, leading to effective but nonspecific interactions with negatively charged cell plasmatic membranes. To reduce the nonspecific internalization of PAMAM dendrimers, their primary amine groups are often modified by acetic or succinic anhydrides, polyethylene glycol derivatives and other compounds. In this work, the role of primary amine groups, which are localized on the surface of doxorubicin-conjugated (Dox) dendrimers, was studied with regard to their intracellular distribution and internalization rates using SKOV3 human ovarian adenocarcinoma cells. It was demonstrated that all Dox-labeled G2-derivatives containing different numbers of acetamide groups synthesized in this work show high rates of cellular uptake at 37°С. As expected, the conjugate carrying the maximum number of primary amine groups demonstrated the highest rates of binding and endocytosis. At the same time, the G2-Dox conjugate containing the maximum number of acetamide groups showed colocalization with LAMP2, a marker of lysosomes and late endosomes, as well as the highest level of cytotoxic activity against SKOV3 cells. We conclude that second-generation PAMAM dendrimers are characterized by varied pathways of internalization and intracellular distribution due to the number of primary amine groups on their surface and, as a consequence, a different surface charge.     

Formulation of anastrozole microparticles as biodegradable anticancer drug carriers

The purpose of this study was to develop poly(d,1-lactic-coglycolic acid) (PLGA)-based anastrozole microparticles for treatment of breast cancer. An emulsion/extraction method was used to prepare anastrozole sustained-release PLGA-based biodegradable microspheres. Gas chromatography with mass spectroscopy detection was used for the quantitation of the drug throughout the studies. Microparticles were formulated and characterized in terms of encapsulation efficiency, particle size distribution, surface morphology, and drug release profile. Preparative variables such as concentrations of stabilizer, drug-polymer ratio polymer viscosity, stirring rate, and ratio of internal to external phases were found to be important factors for the preparation of anastrozole-loaded PLGA microparticles. Fourier transform infrared with attenuated total reflectance (FTIR-ATR) analysis and differential scanning calorimetry (DSC) were employed to determine any interactions between drug and polymer. An attempt was made to fit the data to various dissolution kinetics models for multiparticulate systems, including the zero order, first order, square root of time kinetics, and biphasic models. The FTIR-ATR studies revealed no chemical interaction between the drug and the polymer. DSC results indicated that the anastrozole trapped in the microspheres existed in an amorphous or disordered-crystalline status in the polymer matrix. The highest correlation coefficients were obtained for the Higuchi model, suggesting a diffusion mechanism for the drug release. The results demonstrated that anastrozole microparticles with PLGA could be an alternative delivery method for the long-term treatment of breast cancer. Published: July 21, 2006     

Albumin microspheres as carriers for the antiarthritic drug celecoxib

The present study investigates the preparation of celecoxib-loaded albumin microspheres and the biodistribution of technetium-99m (^99mTc)-labeled celecoxib as well as its microspheres after intravenous administration. Microspheres were prepared using a natural polymer BSA using emulsification chemical cross-linking method. The prepared microspheres were characterized for entrapment efficiency, particle size, and in vitro drug release. Surface morphology was studied by scanning electron microscopy. Biodistribution studies were performed by radiolabeling celecoxib (CS) and its microspheres (CMS) using^99mTc and injecting arthritic rats intravenously. The geometric mean diameter of the microspheres was found to be 5.46 μm. In vitro release studies indicated that the microspheres sustained the release of the drug for }6 days. Radioactivity measured in different organs after intravenous administration of celecoxib solution showed a significant amount of radioactivity in the liver and spleen. In case of celecoxib-loaded microspheres, a significant amount of radioactivity accumulated in the lungs. No significant difference (P>.1) in the radioactivity was observed between the inflamed joint and the noninflamed joint following intravenous injection of^99mTc-CS. However, in case of the microspheres (CMS), the radioactivity present in the inflamed joint was 2.5-fold higher than in the noninflamed joint. The blood kinetic studies revealed that celecoxib-loaded albumin microspheres exhibited prolonged circulation than the celecoxib solution.     

Targeting cancer cells: magnetic nanoparticles as drug carriers

Magnetic drug targeting employing nanoparticles as carriers is a promising cancer treatment avoiding side effects of conventional chemotherapy. We used iron oxide nanoparticles covered by starch derivatives with phosphate groups which bound mitoxantrone as chemotherapeutikum. In this letter we show that a strong magnetic field gradient at the tumour location accumulates the nanoparticles. Electron microscope investigations show that the ferrofluids can be enriched in tumour tissue and tumour cells.     

Development of doxorubicin loading platform based albumin-sporopollenin as drug carrier

Albumin is thought as an drug carrier for doxorubicin (DOX). The binding of doxorubicin to albumin was studied on the surface of sporopolleninin (SP) to produce a new drug system based natural materials. Human serum albumin (HSA) was immobilized on SPIONs in 20 mM Tris buffer, 7.4 of pH. Data showed that binding amount of HSA has been found to be as 285.53 µg to the 25 mg of Sporopolleninin which also bounded 319.76 µM of DOX. Binding of protein and drug to Sp were clarified by SEM, EDX and FT-IR analysis.     

Chitosan oligomers as drug carriers for renal delivery of zidovudine

Chitosan oligomers (COS) are water soluble and can be a potential drug carrier for renal targeting delivery. Zidovudine (AZT), a FDA approved antiretroviral drug, has a very short half life and is eliminated very quickly in human plasma and kidney after administration. In this study, AZT-COS conjugates were prepared and evaluated in terms of renal targeting. The in vitro release of AZT from AZT-COS conjugates was confirmed in mice plasma and renal homogenate. The pharmacokinetics study indicated longer mean retention time of AZT-COS conjugates with the values of about 1.5 h than 0.59 h of AZT. The AZT-COS conjugates were found accumulated in kidney other than heart, liver, spleen, lung and brain after i.v. administration, in line with the evidence of the fluorescence imaging of FITC labeled COS in 12 h. In conclusion, AZT-COS conjugates have the potential to be developed into a renal-targeting drug delivery system.     

Nanostructures by self-assembling peptide amphiphile as potential selective drug carriers

The self-assembling behavior, at physiological pH, of the amphiphile peptide (C18)(2)L5CCK8 in nanostructures is reported. Stable aggregates presenting a critical micellar concentration of 2 x 10(-6) mol kg(-1), and characterized by water exposed CCK8 peptide in beta-sheet conformation, are obtained. Small angle neutron scattering experiments are indicative for a 3D structure with dimensions > or =100 nm. AFM images confirm the presence of nanostructures. Fluorescence experiments indicating the sequestration of pyrene, chosen as drug model, and the anticancer Doxorubicin within the nanostructures are reported.     

An intramolecular cyclization reaction is responsible for the in vivo inefficacy and apparent pH insensitive hydrolysis kinetics of hydrazone carboxylate derivatives of doxorubicin

Contradictory reports concerning the pH sensitive hydrolysis kinetics of certain hydrazone carboxylates of doxorubicin have appeared in this journal (Kaneko et al., Bioconjugate Chem. 1991, 2, 133. Padilla De Jesús et al., Bioconjugate Chem. 2002, 13, 453). Since the pH stability of the drug-carrier linkage in macromolecular prodrugs has a significant bearing on pharmacological efficacy, the hydrolysis kinetics of low molecular weight and polymeric doxorubicin hydrazone carboxylates were therefore reinvestigated. As observed previously, the conjugates readily release native doxorubicin at pH 5. Unexpectedly, in neutral buffer the hydrazone carboxylate conjugates do not release native doxorubicin, but instead rapidly release a doxorubicin derivative substituted at C-9 by 3,6-dihydro-1,3,4-oxadiazin-2-one with first-order kinetics (t(1/2) = 2.5 h). The proposed intramolecular cyclization reaction involving doxorubicin's C-14 hydroxyl and the carboxylate-substituted hydrazone rationalizes the seemingly anomalous hydrolysis kinetics seen for hydrazone carboxylate linked doxorubicin, and provides a possible explanation for the poor antitumor activity exhibited by polymer-doxorubicin conjugates utilizing this specific type of linkage.