J.A. Cadée

In vivo biocompatibility of dextran-based hydrogels.

Dextran-based hydrogels were obtained by polymerization of aqueous solutions of methacrylated dextran (dex-MA) or lactate-hydroxyethyl methacrylate-derivatized dextran (dex-lactate-HEMA). Both nondegradable dex-MA and degradable dex-lactate-HEMA disk-shaped hydrogels, varying in initial water content and degree of substitution (DS, the number of methacrylate groups per 100 glucose units), were implanted subcutaneously in rats. The tissue reaction was evaluated over a period of 6 weeks. The initial foreign-body reaction to the dex-MA hydrogels was characterized by infiltration of granulocytes and macrophages and the formation of fibrin, and exudate, as well as new blood vessels. This reaction depended on the initial water content as well as on the DS of the hydrogel and decreased within 10 days. The mildest tissue response was observed for the gel with the highest water content and intermediate DS. At day 21 all dex-MA hydrogels were surrounded by a fibrous capsule and no toxic effects on the surrounding tissue were found. No signs of degradation were observed. The initial foreign-body reaction to the degradable dex-lactate-HEMA hydrogels was less severe compared with the dex-MA gels. In general, the size of the dex-lactate-HEMA hydrogels increased progressively with time and finally the gels completely dissolved. Degradation of the dex-lactate-HEMA hydrogels was associated with infiltration of macrophages and the formation of giant cells, both of which phagocytosed pieces of the hydrogel. A good correlation between the in vitro and the in vivo degradation time was found. This suggests that extra-cellular degradation is not caused by enzymes but depends only on hydrolysis of the ester and/or carbonate bonds present in the crosslinks of the hydrogels. After 21 days, the degradable hydrogels, as such, could not be retrieved, but accumulation of macrophages and giant cells was observed, both of which contained particles of the gels intracellularly. As for the dex-MA hydrogels, no toxic effects on the surrounding tissue were found. The results presented in this study demonstrate that dextran-based hydrogels can be considered as biocompatible materials, making these hydrogels attractive systems for drug delivery purposes.

Release of recombinant human interleukin-2 from dextran-based hydrogels

In this study, the release of recombinant human interleukin-2 (rhIL-2) from methacrylated dextran (dex-MA) and (lactate-)hydroxyethyl methacrylated dextran (dex-(lactate-)HEMA) hydrogels with varying crosslink density was investigated. Hydrogels derived from dex-MA are stable under physiological conditions (pH 7 and 37 degrees C), whereas dex-HEMA and dex-lactate-HEMA hydrogels degrade due to the presence of hydrolytically sensitive esters in the crosslinks of the gels. The protein release profiles both the non-degradable and degradable dextran-based hydrogels showed that with increasing crosslink density of the gel, the release of rhIL-2 decreases. From dex-MA hydrogels with an initial water content above 70%, the rhIL-2 release followed Fickian diffusion, whereas from gels with an initial water content of 70% or lower the protein was fully entrapped in the hydrogel meshes. In contrast with non-degradable dex-MA hydrogels, degradable dex-lactate-HEMA gels with comparable network characteristics (degree of methacrylate substitution and initial water content) showed an almost zero-order, degradation controlled release of rhIL-2 in a time period of 5-15 days. This paper demonstrates that the release of rhIL-2 from non-degradable dex-MA and degradable dex-lactate-HEMA gels can be modulated by the crosslink density and/or the degradation characteristics of the hydrogel. Importantly, rhIL-2 was mainly released as monomer from the hydrogels and with good retention of its biological activity.

A comparative biocompatibility study of microspheres based on crosslinked dextran or poly(lactic‐co‐glycolic)acid after subcutaneous injection in rats

Microspheres based on methacrylated dextran (dex-MA), dextran derivatized with lactate-hydroxyethyl methacrylate (dex-lactate-HEMA) or derivatized with HEMA (dex-HEMA) were prepared. The microspheres were injected subcutaneously in rats and the effect of the particle size and network characteristics [initial water content and degree of methacrylate substitution (DS)] on the tissue reaction was investigated for 6 weeks. As a control, poly(lactic-co-glycolic)acid (PLGA) microspheres with varying sizes (unsized, smaller than 10 microm, smaller and larger than 20 microm) were injected as well. A mild tissue reaction to the PLGA microspheres was observed, characterized by infiltration of macrophages (MØs) and some granulocytes. Six weeks postinjection, the PLGA microspheres were still present. However, their size was decreased indicating degradation and many spheres had been phagocytosed. The tissue reaction was hardly affected by size differences, except for particles smaller than 10 microm, which induced an extensive tissue reaction. The initial tissue reaction to nondegradable dex-MA microspheres was stronger than towards the PLGA microspheres, but at day 10 the tissue reactions were comparable for both groups. Six weeks postinjection, the dex-MA microspheres were completely phagocytosed, and no signs of degradation were observed. The size and initial water content of dex-MA microspheres hardly affected the tissue response, although less granulocytes were observed for microspheres with higher DS. Slowly degrading dextran microspheres composed of dex-(lactate(1)-)HEMA induced a tissue reaction comparable to the PLGA microspheres. However, degradation of the dex-(lactate(1,3)-)HEMA microspheres was associated with an increased number of MØs and giant cells, both phagocytosing the microspheres and their degradation products. Similar to PLGA, no adverse reactions were observed for the nondegradable dex-MA and degradable dextran microspheres. This study shows that both nondegradable and degradable dextran-based microspheres are well tolerated after subcutaneous injection in rats, which make them interesting candidates as controlled drug delivery systems.

Overexpression of phosphatidylinositol transfer protein alpha in NIH3T3 cells activates a phospholipase A.

In order to investigate the cellular function of the mammalian phosphatidylinositol transfer protein α (PI-TPα), NIH3T3 fibroblast cells were transfected with the cDNA encoding mouse PI-TPα. Two stable cell lines, i.e. SPI6 and SPI8, were isolated, which showed a 2- and 3-fold increase, respectively, in the level of PI-TPα. Overexpression of PI-TPα resulted in a decrease in the duration of the cell cycle from 21 h for the wild type (nontransfected) NIH3T3 (wtNIH3T3) cells and mock-transfected cells to 13–14 h for SPI6 and SPI8 cells. Analysis of exponentially growing cultures by fluorescence-activated cell sorting showed that a shorter G1 phase is mainly responsible for this decrease. The saturation density of the cells increased from 0.20 × 105 cells/cm2 for wtNIH3T3 cells to 0.53 × 105 cells/cm2 for SPI6 and SPI8 cells. However, anchorage-dependent growth was maintained as shown by the inability of the cells to grow in soft agar. Upon equilibrium labeling of the cells withmyo-[3H] inositol, the relative incorporation of radioactivity in the total inositol phosphate fraction was 2–3-fold increased in SPI6 and SPI8 cells when compared with wtNIH3T3 cells. A detailed analysis of the inositol metabolites showed increased levels of glycerophosphoinositol, Ins(1)P, Ins(2)P, and lysophosphatidylinositol (lyso-PtdIns) in SPI8 cells, whereas the levels of phosphatidylinositol (PtdIns) and phosphatidylinositol 4,5-bisphosphate were the same as those in control cells. The addition of PI-TPα to a total lysate ofmyo-[3H]inositol-labeled wtNIH3T3 cells stimulated the formation of lyso-PtdIns. The addition of Ca2+ further increased this formation. Based on these observations, we propose that PI-TPα is involved in the production of lyso-PtdIns by activating a phospholipase A acting on PtdIns. The increased level of lyso-PtdIns that is produced in this reaction could be responsible for the increased growth rate and the partial loss of contact inhibition in SPI8 and SPI6 cells. The addition of growth factors (platelet-derived growth factor, bombesin) to these overexpressers did not activate the phospholipase C-dependent degradation of phosphatidylinositol 4,5-bisphosphate.

Therapeutic efficacy of IL-2-loaded hydrogels in a mouse tumor model.

Interleukin‐2 (IL‐2) is a highly effective anticancer drug if it is applied locally for 5 consecutive days. In most cases this requires 5 invasive treatments, which is not usually acceptable for either the patient or the clinician. For this reason we have developed dextran‐based hydrogels from which the required amount of encapsulated IL‐2 (1–4 × 106 IU of IL‐2) is gradually released during 5–10 days. Initially IL‐2‐containing macroscopic cylinder‐shaped gels (implants), and later IL‐2‐containing injectable microspheres, were developed. These preparations were characterized in vitro, and the therapeutic activity was tested in DBA/2 mice with SL2 lymphosarcoma. The therapy was given to mice with a large and extensively metastasized tumor load (at least 5% of the body weight). If 1–4 × 106 IU of IL‐2 was slowly released from the hydrogels over a period of 5–10 days, the therapeutic effects were very good and comparable to the effects of free IL‐2 injections for 5 consecutive days. In conclusion, dextran‐based hydrogels are promising systems for the controlled release of IL‐2.

Synthesis, characterization of 2-(methacryloyloxy)ethyl-(di-) l-lactate and their application in dextran-based hydrogels

Abstract This paper describes the synthesis and characterization of 2-(methacryloyloxy) ethyl- l -lactate and 2-(methacryloyloxy)ethyl-di- l -lactate. These compounds were obtained by reaction of l -lactide and 2-hydroxyethyl methacrylate (HEMA) at a molar ratio of 1:2 using stannous octoate as catalyst. After a purification step using preparative HPLC, the obtained products were characterized by 1 H-NMR, HPLC and mass spectrometry. 2-(Methacryloyloxy)ethyl-(di-)lactate as well as HEMA were grafted to dextran using 1,1′-carbonyldiimidazole as coupling agent resulting in dex–(lactate) 1,2 –HEMA and dex–HEMA, respectively. Hydrogels were obtained by radical polymerization of aqueous solutions of the methacrylated dextrans. Due to the presence of hydrolytically sensitive groups in the crosslinks, these hydrogels degraded when exposed to an aqueous solution (pH 7.2, 37°C). For hydrogels with a fixed crosslink density, the degradation times were 30, 12 and 6 days for dex–HEMA, dex–lactate–HEMA and dex–(lactate) 2 –HEMA hydrogels, respectively. This demonstrates that the degradation rate and time of dextran hydrogels can be tailored by the length of the spacer in the crosslink of the hydrogels.

Effective cancer therapy with a single injection of interleukin-2 at the site of the tumour

Abstract Mice with a severe metastasized tumour burden can be cured with a single local injection of interleukin-2. Such a treatment can also be effective against ocular squamous cell carcinoma in cows and transmissible venereal tumours in dogs. We did not notice any toxic effects of this treatment.

Oxidation of Recombinant Human Interleukin-2 by Potassium Peroxodisulfate

AbstractPurpose. The oxidation of recombinant human interleukin-2 (rhIL-2) by potassium peroxodisulfate (KPS) with or without N,N,N′,N′-tetramethylethylenediamine (TEMED), which are used for the preparation of dextran-based hydrogels, was investigated. Methods. The oxidation of (derivatives of) methionine, tryptophan, histidine and tyrosine, as well as rhIL-2 was investigated. Both the oxidation kinetics (RP-HPLC) and the nature of the oxidation products (mass spectrometry) were studied as a function of the KPS and TEMED concentration, and the presence of a competitive antioxidant, methionine. Results. Under conditions relevant for the preparation of rhIL-2 loaded hydrogels, only methionine and tryptophan derivatives were susceptible to oxidation by KPS. The oxidation of these compounds was inhibited once TEMED was present, suggesting that the peroxodisulfate anion, rather than the radicals formed in the presence of TEMED, is the oxidative species. KPS only induced oxidation of the four methionines present in rhIL-2, whereas the tryptophan residue remained unaffected. The radicals, formed after KPS decomposition by TEMED, induced some dimerization of rhIL-2. The oxidation of rhIL-2 could be substantially reduced by the addition of methionine, or by pre-incubation of KPS with TEMED. Conclusions. Only the methionine residues in rhIL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions.

IL-2 loaded dextran microspheres with attractive histocompatibility properties for local IL-2 cancer therapy

Biodegradable dextran microspheres (MS) were developed as a slow-release system for interleukin-2 (IL-2) to apply them for local IL-2 therapy of cancer. We describe the tissue reactions induced by these MS without or with IL-2 in rats. Dextran MS stain bright red-purple with the periodic acid Schiff (PAS), visualising the exact spot of IL-2 release and its relation to the histological reaction pattern. Subcutaneously injected MS always form a well-circumscribed deposit. In the first 2 days there is a PMN inflammation within the MS-deposit, but the surroundings show only a scanty inflammatory reaction. The PMN reaction is replaced by an abundant macrophage reaction in particular in the MS-deposit. At day 21 a fibrous capsule of about 50 mum surrounds the deposit. The effect of IL-2 administered in its free form is mainly vascular, with vascular dilatation, vascular leakage and oedema. It is remarkable that lymphocytes are present in the injection area already at day 2. When IL-2 releasing MS were used, the various reactions induced by IL-2 and MS were amplified leading to local necrosis. We conclude that neither placebo MS nor IL-2 leads to necrosis after subcutaneous injection in rats. In contrast, when IL-2 was released from MS, then massive necrosis was induced. This might be due to increased phagocytosis or changes in the micro-niche due to the release of humoral factors by the infiltrating cells. This is probably fortuitous for local IL-2 therapy of cancer, as massive necrosis of tumour cells can be expected to lead to an increased antitumour reaction.

Molecular Design of Biodegradable Dextran Hydrogels for the Controlled Release of Proteins

A biodegradable and biocompatible dextran hydrogel system has been developed of which the degradation time can be tailored between 2 days and 3 months. This chapter shows that hydrogels based on crosslinked dextrans have unique properties as protein releasing matrices. Both the release pattern (first-order, zero-order or biphasic) as well as the duration of the release can be controlled by an appropriate selection of the characteristics and the geometry of the hydrogel.