Liposomal drug formulations in the treatment of rheumatoid arthritis.

Liposomes have been extensively investigated as drug delivery systems in the treatment of rheumatoid arthritis (RA). Low bioavailability, high clearance rates and limited selectivity of several important drugs used for RA treatment require high and frequent dosing to achieve sufficient therapeutic efficacy. However, high doses also increase the risk for systemic side effects. The use of liposomes as drug carriers may increase the therapeutic index of these antirheumatic drugs. Liposomal physicochemical properties can be changed to optimize penetration through biological barriers and retention at the site of administration, and to prevent premature degradation and toxicity to nontarget tissues. Optimal liposomal properties depend on the administration route: large-sized liposomes show good retention upon local injection, small-sized liposomes are better suited to achieve passive targeting. PEGylation reduces the uptake of the liposomes by liver and spleen, and increases the circulation time, resulting in increased localization at the inflamed site due to the enhanced permeability and retention (EPR) effect. Additionally liposomal surfaces can be modified to achieve selective delivery of the encapsulated drug to specific target cells in RA. This review gives an overview of liposomal drug formulations studied in a preclinical setting as well as in clinical practice. It covers the use of liposomes for existing antirheumatic drugs as well as for new possible treatment strategies for RA. Both local administration of liposomal depot formulations and intravenous administration of passively and actively targeted liposomes are reviewed.

Effect of particle size and charge on the network properties of microsphere-based hydrogels.

This work describes the tailorability of the network properties of self-assembling hydrogels, based on ionic crosslinking between dextran microspheres. Copolymerization of hydroxyethyl methacrylate-derivatized dextran (dex-HEMA), emulsified in an aqueous poly(ethylene glycol) (PEG) solution, with methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) resulted in negatively or positively charged microspheres, respectively, at physiological pH. The monomer/HEMA ratio ranged between 6 and 57, resulting in microspheres with zeta (zeta)-potentials from -6 to -34mV and +3 to +23mV, for the monomers MAA and DMAEMA, respectively. By altering the emulsification procedure, microsphere batches with various sizes and size distributions were obtained. The aim of the research was to assess the effect of particle size (distribution) and charge on the network properties of the macroscopic hydrogels. The ability to tailor the mechanical properties such as strength and elasticity increases the potential of the hydrogels to be used in a variety of pharmaceutical applications. Additionally, the injectability of these self-assembling hydrogels was investigated. Injectability is an important feature of drug delivery systems, since it allows avoiding surgery. Rheological analysis showed that an increasing surface charge of the microspheres led to stronger hydrogels. Relatively small microspheres (7microm) with a narrow size distribution (99% smaller than 14microm) gave rise to stronger hydrogels when compared to larger microspheres of 20microm with a broad distribution (99% smaller than 50microm). When small microspheres were combined with large microspheres of opposite charge, it was found that the strongest gels were obtained with 75% small and 25% large microspheres, instead of equal amounts (50/50) of positively and negatively charged microspheres. Computer modeling confirmed these findings and showed that the most favorable composition, related to the lowest potential energy, comprised of 75% small microspheres. Taking both charge and size effects into account, the storage moduli (G') of the almost fully elastic hydrogels could be tailored from 400 to 30,000Pa. Injectability tests showed that hydrogels (G' up to 4000Pa) composed of equal amounts of oppositely charged microspheres (-7 and +6mV, average particle size 7microm) could be injected through 25G needles using a static load of 15N, an ISO accepted value. In conclusion, a variety of options to control the network properties of macroscopic hydrogels are provided, related to the charge and particle size of the composing dextran microspheres. Furthermore, it is shown that the hydrogels are injectable, making them attractive candidates for a diversity of pharmaceutical applications.

In situ gelling hydrogels for pharmaceutical and biomedical applications.

Since Wichterle et al. introduced hydrogels as novel materials possibly suitable for a variety of biomedical applications, hydrogel research has become a fast-developing and exciting research field. The soft and hydrophilic nature of hydrogels makes them particularly suitable as protein delivery system or as cell-entrapping scaffold in tissue engineering. Traditional hydrogels were formed by chemical crosslinking of water-soluble polymers or by polymerization (of mixtures) of water-soluble monomers. Because of incompatibility of these crosslinking methods with fragile molecules like pharmaceutical proteins and living cells, in recent years research interest has been focused on hydrogels that gel spontaneously under physiological conditions. In these systems, hydrogel formation occurs in situ, at the site of injection, without the aid of potentially toxic or denaturizing crosslinking agents. This review provides an overview of in situ gelling systems and their potential in biomedical applications. Both photopolymerizable as well as self-assembling hydrogels, based on either chemical crosslinks or physical interactions will be addressed.

Macroscopic hydrogels by self-assembly of oligolactate-grafted dextran microspheres.

A novel approach is presented to create self-assembling hydrogels. Microspheres based on cross-linked dextran were chemically modified with L- or D-oligolactate chains. Successful grafting of the particles was confirmed by Fourier transform infrared (FT-IR) and Raman and X-ray photoelectron spectroscopy (XPS). Rheological analysis of aqueous dispersions of oligolactate-grafted microspheres demonstrated that hydrophobic interactions between oligolactate chains on the surface of various microspheres resulted in the formation of an almost fully elastic gel. A mixture of microspheres substituted with L- or D-oligolactates of opposite chirality resulted in gels with highest strength, likely due to stereocomplexation between the enantiomers. The network properties could be modulated by varying the solid content of the gel, the DS (i.e., number of lactate grafts per 100 glucopyranose units) and the DP (i.e., degree of polymerization) of the oligolactate grafts. Protein loading of the hydrogels could be achieved by simply mixing the microspheres with protein solution. Release experiments showed a continuous release of the entrapped lysozyme, with 50% released after 5 days and full preservation of its enzymatic activity. The biocompatible nature of the material, the protein-friendly self-assembly of the hydrogel and the possibility to tailor the gel properties, makes this hydrogel system an attractive candidate for pharmaceutical and biomedical applications.

In vitro and in vivo protein delivery from in situ forming poly(ethylene glycol)-poly(lactide) hydrogels.

Previous studies have shown that stereocomplexed hydrogels are rapidly formed in situ by mixing aqueous solutions of eight-arm poly(ethylene glycol)-poly(L-lactide) and poly(ethylene glycol)-poly(D-lactide) star block copolymers (denoted as PEG-(PLLA)(8) and PEG-(PDLA)(8), respectively). In this study, in vitro and in vivo protein release from stereocomplexed hydrogels was investigated. These hydrogels were fully degradable under physiological conditions. Proteins could be easily loaded into the stereocomplexed hydrogels by mixing protein containing aqueous solutions of PEG-(PLLA)(8) and PEG-(PDLA)(8) copolymers. The release of the relatively small protein lysozyme (d(h)=4.1 nm) followed first order kinetics and approximately 90% was released in 10 days. Bacteria lysis experiments showed that the released lysozyme had retained its activity. The relatively large protein IgG (d(h)=10.7 nm) could be released from stereocomplexed hydrogels with nearly zero order kinetics, wherein up to 50% was released in 16 days. The in vitro release of the therapeutic protein rhIL-2 from stereocomplexed hydrogels also showed nearly zero order kinetics, wherein up to 45% was released in 7 days. The therapeutic efficacy of stereocomplexed hydrogels loaded with 1x10(6) IU of rhIL-2 was studied using SL2-lymphoma bearing DBA/2 mice. The PEG-(PLLA)(8)/PEG-(PDLA)(8)/rhIL-2 mixture could be easily injected intratumorally. The released rhIL-2 was therapeutically effective as the tumor size was reduced and the cure rate was 30%, whereas no therapeutic effect was achieved when no rhIL-2 was given. However, the cure rate of rhIL-2 loaded stereocomplexed hydrogels was lower, though not statistically significant, compared to that of a single injection with 1x10(6) IU of free rhIL-2 at the start of the therapy (cure rate=70%). The therapeutic effect of rhIL-2 loaded stereocomplexed hydrogels was retarded for approximately 1-2 weeks compared to free rhIL-2, most likely due to a slow, constant release of rhIL-2 from the hydrogels.

Biodegradable dextran hydrogels for protein delivery applications.

The rapid development of protein-based pharmaceuticals over recent decades has tremendously increased the need for suitable delivery systems, guaranteeing a safe and controlled delivery of proteinacious drugs. Hydrogels offer good opportunities as protein delivery systems or tissue engineering scaffolds owing to an inherent biocompatibility. Their hydrophilic, soft and rubbery nature ensures minimal tissue irritation and a low tendency of cells and proteins to adhere to the hydrogel surface. A variety of both natural and synthetic polymers have been used for the design of hydrogels, in which network formation is established by chemical or physical crosslinking. This review introduces the general features of hydrogels and focuses on dextran hydrogels in particular. Chemically and physically crosslinked systems are described and their potential suitability as protein delivery systems, as well as tissue engineering scaffolds are discussed. Special attention is given to network properties, protein delivery, degradation behavior and biocompatibility.

Degradation behavior of dextran hydrogels composed of positively and negatively charged microspheres.

This paper reports on the degradation behavior of in situ gelling hydrogel matrices composed of positively and negatively charged dextran microspheres. Rheological analysis showed that, once the individual microspheres started to degrade, the hydrogel changed from a mainly elastic to a viscoelastic network. It was shown with gels composed of equal amounts of cationic and anionic microspheres, that both a higher crosslink density of the particles and a decrease in water content of the hydrogels resulted in a slower degradation, ranging from 65 to 140 days. Dispersions containing cationic, neutral or anionic microspheres completely degraded within 30, 55 or 120 days, respectively. The microspheres were loaded with rhodamine-B-dextran and degradation was studied with confocal microscopy and fluorescence spectroscopy. After a lag time of 3 days rhodamine-B-dextran started to release from the positive microspheres with a 50% release after 16 days. In contrast, release of rhodamine-B-dextran from the negative microspheres started after 10 days with a 50% release after 36 days. The faster degradation of the positively charged microspheres as compared to the negatively charged microspheres is attributed to stabilization of the transition state in the hydrolysis process by the protonated tertiary amine groups present in the cationic microspheres. On the other hand, the presence of negatively charged groups causes repulsion of hydroxyl anions resulting in a slower degradation. Combining the oppositely charged microspheres in different ratios makes it possible to tailor the network properties and the degradation behavior of these hydrogels, making them suitable for various applications in drug delivery and tissue engineering.

Mobility of model proteins in hydrogels composed of oppositely charged dextran microspheres studied by protein release and fluorescence recovery after photobleaching.

In this paper, the release of proteins from a novel self-gelling hydrogel based on biodegradable dextran microspheres is investigated. The protein-loaded macroscopic gels are obtained by hydration of mixtures of oppositely charged hydroxyethyl methacrylate-derivatized dextran microspheres with a protein solution. In media of low ionic strength (100 mM Hepes pH 7.0) it was found that the release of the entrapped model proteins (lysozyme, BSA and IgG) was slower than in saline (150 mM NaCl, 100 mM Hepes pH 7.0). The reason behind this observation is that substantial adsorption of the proteins onto the microspheres' surface and/or absorption in the microspheres takes place. Confocal images showed that independent of their crosslink density the microspheres are impermeable for BSA and IgG. BSA, bearing a negative charge at neutral pH, was adsorbed onto the surface of positively charged microspheres. Lysozyme, which is positively charged at neutral pH, was able to penetrate into the negatively charged microspheres. In saline, the gels showed continuous release of the different proteins for 25 to 60 days. Importantly, lysozyme was quantitatively and with full preservation of its enzymatic activity released in about 25 days. This emphasizes the protein friendly technology to prepare the protein-loaded gels. Mathematical modeling revealed that protein release followed Fick's second law, indicating that the systems are primarily diffusion controlled. These results show that these hydrogels are very suitable as injectable matrix for diffusion-controlled delivery of pharmaceutically active proteins.

Self-gelling hydrogels based on oppositely charged dextran microspheres.

This paper presents a novel self-gelling hydrogel potentially suitable for controlled drug delivery and tissue engineering. The macroscopic gels are obtained by mixing dispersions of oppositely charged crosslinked dextran microspheres. These microspheres in turn were prepared by crosslinking of dextran derivatized with hydroxyethyl methacrylate emulsified in an aqueous poly(ethylene glycol) solution. Negatively or positively charged microspheres were obtained by addition of methacrylic acid (MAA) or dimethylaminoethyl methacrylate (DMAEMA) to the polymerization mixture. Rheological analysis showed that instantaneous gelation occurred when equal volumes of oppositely charged microspheres, dispersed in buffer solutions of pH 7, were mixed. The shear modulus of the networks could be tailored from 30 to 6500 Pa by varying the water content of the system. Moreover, controlled strain and creep experiments showed that the formed networks were mainly elastic. Importantly for application of these systems, e.g. as controlled matrix of pharmaceutically active proteins, it was demonstrated that the hydrogel system has a reversible yield point, meaning that above a certain applied stress, the system starts to flow, whereas when the stress is removed, gel formation occurred. Further it was shown that the network structure could be broken by either a low pH or a high ionic strength of the medium. This demonstrates that the networks, formed at pH 7 and at low ionic strength, are held together by ionic interactions between the oppositely charged dextran microspheres. This system holds promise as injectable gels that are suitable for drug delivery and tissue engineering applications.

In situ crosslinked biodegradable hydrogels loaded with IL-2 are effective tools for local IL-2 therapy.

We investigated the therapeutic efficacy of recombinant human interleukin-2 (rhIL-2)-loaded, in situ gelling, physically crosslinked dextran hydrogels, locally applied to SL2 lymphoma in mice. The physical crosslinking was established by stereocomplex formation between d-lactic acid oligomers and l-lactic acid oligomers grafted separately to dextrans. The stereocomplex hydrogel as described in our manuscript has several favourable characteristics, which enables its use as system for the controlled release of pharmaceutically active proteins. Firstly, the hydrogel system is a physically crosslinked system. In physically crosslinked gels, the use of chemical crosslinking agents is avoided. Such agents can potentially inactivate the protein and can covalently link the protein to the hydrogel network. Secondly, the hydrogel formation takes place at room temperature and physiological pH, and, importantly, in an all-aqueous environment. All factors are important to preserve the three-dimensional structure, and thus the biological activity, of the protein to be entrapped and released from the gels. Thirdly, the gel formation does not occur instantaneously. This means that a liquid formulation can be injected which solidifies after injection (in situ gel formation is possible). Fourthly, no pH drop during degradation is expected during degradation. As a control, free rhIL-2 was administered locally in either a single injection or at five consecutive days. All mice received the same total dose of rhIL-2. The rhIL-2-loaded hydrogels released most IL-2 over a period of about 5 days. The biocompatibility and biodegradability of the gels were excellent, as there were no acute or chronic inflammatory reaction and as the gels were replaced completely by fibroblasts after 15 days. The therapeutic efficacy of rhIL-2-loaded in situ gelled hydrogels is very good, as was demonstrated in DBA/2 mice bearing SL2. The therapeutic effect of a single application of gels loaded with 1 x 10(6) IU rhIL-2 is at least comparable to the therapeutic effect of injection of an equal dose of free rhIL-2. All mice cured with rhIL-2-loaded hydrogels survived a subsequent challenge, rejecting 10(6) intraperitoneal (i.p.) injected SL2 cells. In conclusion, this study demonstrates that in situ gelling, physically crosslinked dextran hydrogels slowly release encapsulated rhIL-2 in such a way that it is intact and biologically and therapeutically active. These hydrogels may greatly enhance the clinical applicability of rhIL-2 immunotherapy as only a single treatment is required and as these hydrogels are completely biodegradable.