Bactericidal coating to prevent early and delayed implant-related infections.

The occurrence of an implant-associated infection (IAI) with the formation of a persisting bacterial biofilm remains a major risk following orthopedic biomaterial implantation. Yet, progress in the fabrication of tunable and durable implant coatings with sufficient bactericidal activity to prevent IAI has been limited. Here, an electrospun composite coating was optimized for the combinatorial and sustained delivery of antibiotics. Antibiotics-laden poly(ε-caprolactone) (PCL) and poly`1q`(lactic-co glycolic acid) (PLGA) nanofibers were electrospun onto lattice structured titanium (Ti) implants. In order to achieve tunable and independent delivery of vancomycin (Van) and rifampicin (Rif), we investigated the influence of the specific drug-polymer interaction and the nanofiber coating composition on the drug release profile and durability of the polymer-Ti interface. We found that a bi-layered nanofiber structure, produced by electrospinning of an inner layer of [PCL/Van] and an outer layer of [PLGA/Rif], yielded the optimal combinatorial drug release profile. This resulted in markedly enhanced bactericidal activity against planktonic and adherent Staphylococcus aureus for 6 weeks as compared to single drug delivery. Moreover, after 6 weeks, synergistic bacterial killing was observed as a result of sustained Van and Rif release. The application of a nanofiber-filled lattice structure successfully prevented the delamination of the multi-layer coating after press-fit cadaveric bone implantation. This new lattice design, in conjunction with the multi-layer nanofiber structure, can be applied to develop tunable and durable coatings for various metallic implantable devices. This is particularly appealing to tune the release of multiple antimicrobial agents over a period of weeks to prevent early and delayed onset IAI.

Thermotropic properties of phosphatidylcholine nanodiscs bounded by styrene-maleic acid copolymers.

Styrene-maleic acid copolymers (SMA) have been gaining interest in the field of membrane research due to their ability to solubilize membranes into nanodics. The SMA molecules act as an amphipathic belt that surrounds the nanodiscs, whereby the hydrophobic styrene moieties can insert in between the lipid acyl chains. Here we used SMA variants with different styrene-to-maleic acid ratio (i.e. 2:1, 3:1 and 4:1) to investigate how lipid packing in the nanodiscs is affected by the presence of the polymers and how it depends on polymer composition. This was done by analyzing the thermotropic properties of a series of saturated phosphatidylcholines in nanodiscs using laurdan fluorescence and differential scanning calorimetry. In all cases it was found that the temperature of the main phase transition (Tm) of the lipids in the nanodiscs is downshifted and that its cooperativity is strongly reduced as compared to the situation in vesicles. These effects were least pronounced for lipids in nanodiscs bounded by SMA 2:1. Unexpected trends were observed for the calorimetric enthalpy of the transition, suggesting that the polymer itself contributes, possibly by rearranging around the nanodiscs when the lipids adopt the fluid phase. Finally, distinct differences in morphology were observed for nanodiscs at relatively high polymer concentrations, depending on the SMA variant used. Overall, the results suggest that the extent of preservation of native thermodynamic properties of the lipids as well as the stability of the nanodiscs at high polymer concentrations is better for SMA 2:1 than for the other SMA variants.

The effect of lauryl capping group on protein release and degradation of poly(D,L-lactic-co-glycolic acid) particles.

The aim of this study was to investigate the effect of a specific and frequently used end group (lauryl alcohol) on the protein release and degradation kinetics of poly(DL-lactic-co-glycolic acid) particles of different sizes. Lauryl-capped PLGA and uncapped PLGA (referred to as PLGA-capped and PLGA-COOH, respectively) particles (0.3, 1 and 20 µm) were prepared by a double emulsion solvent evaporation technique. Bovine serum albumin (BSA) was used as a model protein for release studies. During degradation (PBS buffer, pH7.4 at 37°C), a slower dry mass loss was observed for 0.3 µm particles than for particles of 1 and 20 µm. It was further shown that PLGA-capped particles showed slower mass loss likely due to its more hydrophobic nature. It was found that the ester bond hydrolysis rate was substantially slower for PLGA-capped particles and that the rate increased with particle size. Particles showed enrichment in lactic acid content (and thus a decrease in glycolic acid content) in time, and interestingly PLGA-capped particles showed also an enrichment of the lauryl alcohol content. No difference was observed in degradation kinetics between BSA loaded and blank particles. Independent of size, PLGA-COOH based particles showed, after a small burst, a sustained and nearly complete release of BSA during 60-80 days. On the other hand, particles based on PLGA-capped showed a much slower release and exhibited incomplete release, accompanied by the presence of an insoluble residue remaining even after 180 days. FTIR analysis of this residue showed that it contained both polymer and protein. Considering the polymer enrichment in lauryl alcohol, the incomplete release observed for PLGA-capped is likely attributed to interactions between the protein and the lauryl end group. In conclusion, since PLGA-COOH, in contrast to the capped derivative, shows complete degradation as well as quantitative release of an entrapped protein, this polymer is preferred for the design of protein formulations.

Preparation and characterization of poly(lactic-co-glycolic acid) microspheres loaded with a labile antiparkinson prodrug.

L-dopa-α-lipoic acid (LD-LA) is a new multifunctional prodrug for the treatment of Parkinson's disease. In human plasma, LD-LA catechol esters and amide bonds are chemically and enzymatically cleaved, respectively, resulting in a half-life time of about fifty minutes. In the present work, the unstable LD-LA was entrapped into biodegradable poly(lactic-co-glycolic acid) (PLGA) microspheres designed as depot systems to protect this prodrug against degradation and to obtain a sustained release of the intact compound. The microspheres were prepared by an oil-in-water emulsion/solvent evaporation technique and the effect of formulation and processing parameters (polymer concentration in the organic solvent, volumes ratio of the phases, rate of the organic solvent evaporation) on microspheres characteristics (size, loading, morphology, release) was investigated. Also emphasis was given on the stability of the drug before and after release as well as on the underlying mass transport mechanisms controlling LD-LA release. Interestingly, when encapsulated in appropriate conditions into PLGA microspheres, the labile prodrug was stabilized and released via Fickian diffusion up to more than one week.

Photopolymerized thermosensitive hydrogels for tailorable diffusion-controlled protein delivery.

In this paper the possibility to tailor degradation and protein release behavior of photopolymerized thermosensitive hydrogels is studied. The hydrogels consist of ABA triblock copolymer, in which the thermosensitive A-blocks are methacrylated poly(N-(2-hydroxypropyl)methacrylamide lactate)s and the B-block is poly(ethylene glycol) with molecular weight of 10 kDa. These hydrogels are prepared by using a combination of physical and chemical cross-linking methods. When a solution of a thermosensitive methacrylated p(HPMAm-lac)-PEG-p(HPMAm-lac) is heated above its cloud point a viscoelastic material is obtained, which can be stabilized by introducing covalent cross-links by photopolymerization. By varying the polymer concentration, hydrogels with different mechanical properties are formed, of which the cross-linking density, mesh size, swelling and degradation behavior can be tuned. It was demonstrated that the release rate of three model proteins (lysozyme, BSA and IgG, with hydrodynamic diameters ranging from 4.1 to 10.7 nm) depended on the protein size and hydrogel molecular weight between cross-links and was governed by the Fickian diffusion. Importantly, the encapsulated proteins were quantitatively released and the secondary structure and the enzymatic activity of lysozyme were fully preserved demonstrating the protein friendly nature of the studied delivery system.

Preparation and characterization of protein loaded microspheres based on a hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid).

The purpose of this study was to investigate the suitability of a novel hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid) (PLHMGA), as controlled release system for pharmaceutical proteins. Dextran Blue (as a macromolecular model compound) and lysozyme-loaded PLHMGA and PLGA (control formulation) microspheres were prepared by a solvent evaporation technique. The Dextran Blue and lysozyme loaded PLHMGA microspheres prepared with 10% polymer solution showed, because of a high porosity, a high burst release (35-75%) and the remaining content was released in a sustained manner for 15-20 days. The microspheres prepared with 15 and 20% polymer solution had a lower porosity and showed a pulsed release after day 8 and in 27 days they released more than 90% of Blue Dextran. The release of lysozyme was incomplete, likely due to aggregation of part of the encapsulated protein. Spectroscopic analysis of the released lysozyme indicated fully preserved secondary/tertiary structure and an enzyme activity assay showed that the specific activity of the released protein was maintained. An in vitro degradation study showed that the release of Blue Dextran and lysozyme is essentially controlled by the degradation of the microspheres. This study shows that microspheres made of the hydroxylated aliphatic polyester, poly(lactic-co-hydroxymethyl glycolic acid), are promising systems for the controlled release of pharmaceutical proteins.

Neutron activation of holmium poly(L-lactic acid) microspheres for hepatic arterial radio-embolization: a validation study.

Poly(L-lactic acid) microspheres loaded with holmium-166 acetylacetonate (166Ho-PLLA-MS) are a novel microdevice for intra-arterial radio-embolization in patients with unresectable liver malignancies. The neutron activation in a nuclear reactor, in particular the gamma heating, damages the 166Ho-PLLA-MS. The degree of damage is dependent on the irradiation characteristics and irradiation time in a particular reactor facility. The aim of this study was to standardize and objectively validate the activation procedure in a particular reactor. The methods included light- and scanning electron microscopy (SEM), particle size analysis, differential scanning calorimetry, viscometry, thermal neutron flux measurements and energy deposition calculations. Seven hours-neutron irradiation results in sufficient specific activity of the 166Ho-PLLA-MS while structural integrity is preserved. Neutron flux measurements and energy deposition calculations are required in the screening of other nuclear reactors. For the evaluation of microsphere quality, light microscopy, SEM and particle size analysis are appropriate techniques.

Degradable PEG-folate coated poly(DMAEA-co-BA)phosphazene-based polyplexes exhibit receptor-specific gene expression.

A new cationic biodegradable polyphosphazene was developed, bearing both pendant primary and tertiary amine side groups, poly(2-dimethylaminoethylamine-co-diaminobutane)phosphazene (poly(DMAEA-co-BA)phosphazene). PEG and PEG-folate were coupled to polyplexes based on this poly(DMAEA-co-BA)phosphazene, leading to small (size 100 and 120nm, respectively) and almost neutral particles. In vitro tissue culture experiments showed a low cytotoxicity of both uncoated and coated polyplexes. However, the PEG coated polyplexes showed a 2-fold lower transfection activity in OVCAR 3 cells as compared to the uncoated polyplexes. On the other hand, the PEG-folate coated polyplexes had a 3-fold higher transfection than the PEGylated polyplexes. When free folate was added to the transfection medium, only the transfection activity of the targeted polyplexes was reduced, indicating internalization of the targeted PEG polyplexes via the folate receptor. Confocal laser scanning microscopy confirmed a lower binding and uptake of the PEGylated polyplexes by OVCAR-3 cells when compared to uncoated and folate-PEGylated polyplexes. While uncoated polyplexes induced aggregation of erythrocytes at polymer concentrations of 0.09microg/mL, the PEGylated systems could be incubated at ten times higher concentration before aggregation occurred indicating excellent shielding of the surface charge of the polyplexes by grafting of PEG. In conclusion, the targeted delivery of poly(DMAEA-co-BA)phosphazene bases polyplexes and their improved compatibility with erythrocytes makes them interesting for in vivo applications.

Effect of excipients on the encapsulation efficiency and release of human growth hormone from dextran microspheres.

The possibility was investigated to modulate the encapsulation efficiency and release of human growth hormone (hGH) from hydroxyl ethyl methacrylated dextran (dex-HEMA) hydrogel microspheres by using excipients. Microspheres were prepared by polymerization of dex-HEMA in an aqueous two-phase system of this polymer and PEG with or without excipients (Tween 80, pluronic F68, sucrose, NaCl, urea or methionine). High hGH encapsulation efficiencies (50-70%) were obtained for microspheres prepared without excipients and with Tween 80, NaCl or methionine. Substantially lower encapsulation efficiencies (27% and 19%, respectively) were obtained for microspheres prepared in the presence of sucrose and urea, which was attributed to the more favoured partitioning of hGH over the PEG-phase due to higher hydrophobicity of the (partly) denatured hGH. Likely, differences in precipitate size of the encapsulated hGH resulted in different release profiles between microspheres prepared without excipients (biphasic release: 2 days delay time followed by 6 days release) and the release profile for microspheres prepared with Tween 80, pluronic F68, sucrose, NaCl and urea (release over a period of 6-8 days (without a delay time)). Microspheres prepared with methionine showed a concentration-dependent delay time varying from 0 to 2 days followed by almost zero-order release over 6 days, attributed to the effect of methionine on the polymerization of dex-HEMA. Especially, Tween 80 and methionine are attractive excipients since hGH was encapsulated in high yield (60-70%) and the protein was released from the microspheres mainly in its monomeric form without a delay time and with an almost zero-order release over 6-8 days.

Pharmaceutical development of a parenteral lyophilised dosage form for the novel anticancer agent C1311.

C1311 (5-[[2-(diethylamino)ethyl]amino]-8-hydroxyimidazo [4,5,1-de]-acridin-6-one-dihydrochloride trihydrate) is the lead compound from the group of imidazoacridinones, a novel group of rationally designed anticancer agents. C1311 shows significant cytotoxic activity in vitro and in vivo toward a range of colon tumours. The aim of the present study is to develop a sterile and stable, injectable pharmaceutical product for C1311 to be used in phase I clinical trials. C1311 drug substance was structurally and analytically characterised by chromatographic, spectrometric, and diffraction techniques. C1311 was freely soluble in water, and its stability was investigated in several liquid and lyophilised formulations with or without the use of buffering, tonicity, and bulking agents. The final product, containing 100 mg/vial C1311 (as anhydrous free base), was stable for at least 3 months under accelerated storage conditions and at the designated long-term storage condition of 5 +/- 3 degrees C in the dark. The drug is currently used in phase I clinical trials.

Homogeneous nucleation of water in mesoporous zeolite cavities.

In this paper, we show that water inside mesoporous cavities in zeolites can be supercooled to ca. -40 degrees C at which point homogeneous nucleation of the water takes place. The fundamental phenomena observed here are similar to those reported earlier in for example emulsion droplets or droplets in the vapor phase. However, as these zeolite materials are widely available, they may provide an easily accessible source for studies of supercooled liquids in confinements. Next to this, it is now possible to discriminate with thermoporometry between mesoporous cavities inside the zeolite crystals, in which homogeneous nucleation takes place, and mesopores that are connected to the external surface in which heterogeneous nucleation takes place.

Pharmaceutical development of a parenteral lyophilized formulation of the investigational polymer-conjugated platinum anticancer agent AP 5280.

AP 5280 is a novel polymer-conjugated platinum anticancer agent showing promising in vitro and in vivo activity against solid tumors. The aim of this study was to develop a parenteral pharmaceutical dosage form for phase I clinical trials. AP 5280 drug substance was characterized by using a wide range of analytical techniques and showed excellent solubility in water. However, as aqueous solutions of AP 5280 proved to be labile upon sterilization by moist heat, it was decided to develop a lyophilized dosage form. Initially, glass vials were used as primary packaging, but this led to a high breakage rate, which could be completely prevented by the use of CZ resin vials. Stability studies to date show that the lyophilized product in glass vials is stable for at least 12 months when stored at 2-8 degrees C in the dark and the lyophilized product in CZ resin vials is stable for at least 6 months under these conditions. Photostability testing revealed photolability of AP 5280 drug substance and lyophilized product in both types of primary container, necessitating storage in the dark. The first clinical experiences indicate that the proposed formulation is fully applicable for use in the clinical setting.

Pharmaceutical development of a parenteral lyophilized formulation of the antimetastatic ruthenium complex NAMI-A.

This paper describes the development of a stable pharmaceutical dosage form for NAMI-A, a novel antimetastatic ruthenium complex, for Phase I testing. NAMI-A drug substance was characterized using several spectrometric and chromatographic techniques. In preformulation studies, it was found that NAMI-A in aqueous solution was not stable enough to allow sterilization by moist heat. The effect of several excipients on the stability of the formulation solution was investigated. None of them provided sufficient stability to allow long-term storage of an aqueous solution of NAMI-A. Therefore, a lyophilized product was developed. Five different formulations were prepared and subjected to thermogravimetric (TG) analysis and stability studies at various conditions for 1 year. Minimal degradation during the production process is achieved with a formulation solution of pH 3-4. Of the acids tested, only hydrochloric acid (HCl 0.1 mM) both stabilized the formulation solution and was compatible with the lyophilized product. This product was stable for at least 1 year when stored at -20 degrees C, 25 degrees C/60% relative humidity (RH) and 40 degrees C/75% RH, and was also photostable.

Influence of neutron irradiation on holmium acetylacetonate loaded poly(L-lactic acid) microspheres.

Holmium-loaded microspheres are useful systems in radio-embolization therapy of liver metastases. For administration to a patient, the holmium-loaded microspheres have to be irradiated in a nuclear reactor to become radioactive. In this paper. the influence of neutron irradiation on poly(L-lactic acid) (PLLA) microspheres and films, with or without holmium acetylacetonate (HoAcAc), is investigated, in particular using differential scanning calorimetry (MDSC), scanning electron microscopy, gel permeation chromatography (GPC), infrared spectroscopy, and X-ray diffraction. After irradiation of the microspheres, only minor surface changes were seen using scanning electron microscopy, and the holmium complex remained immobilized in the polymer matrix as reflected by a relatively small release of this complex. GPC and MDSC measurements showed a decrease in molecular weight and crystallinity of the PLLA, respectively, which can be ascribed to radiation induced chain scission. Irradiation of the HoAcAc loaded PLLA matrices resulted in evaporation of the non-coordinated and one coordinated water molecule of the HoAcAc complex, as evidenced by MDSC and X-ray diffraction analysis. Infrared spectroscopy indicated that some degradation of the acetylacetonate anion occurred after irradiation. Although some radiation induced damage of both the PLLA matrix and the embedded HoAcAc-complex occurs, the microspheres retain their favourable properties (no marginal release of Ho, preservation of the microsphere size), which make these systems interesting candidates for the treatment of tumours by radio-embolization.

Oxidation of recombinant human interleukin-2 by potassium peroxodisulfate.

PURPOSE: The oxidation of recombinant human interleukin-2 (rhlL-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 rhlL-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 rhlL-2 are oxidized by KPS. The extent of oxidation can be minimized by a proper selection of the reaction conditions.

Characterization of poly(L-lactic acid) microspheres loaded with holmium acetylacetonate.

Holmium-loaded PLLA microspheres are useful systems in radioembolization therapy of liver metastases because of their low density, biodegradability and favourable radiation characteristics. Neutron activated Ho-loaded microspheres showed a surprisingly low release of the relatively small holmium complex. In this paper factors responsible for this behaviour are investigated, in particular by the use of differential scanning calorimetry, scanning electron microscopy, infrared spectroscopy and X-ray diffraction. The holmium complex is soluble in PLLA up to 8% in films and 17% in microspheres. Interactions between carbonyl groups of PLLA, and the Ho-ion in the HoAcAc complex, explain very satisfactorily the high stability of holmium-loaded microspheres.

Effect of molecular weight and glass transition on relaxation and release behaviour of poly(DL-lactic acid) tablets.

Different molecular weight grades of poly(DL-lactic acid) were applied as release controlling excipients in tablets for oral drug administration. The role of molecular weight and glass transition in the mechanism of water-induced volume expansion and drug release of PDLA tablets was investigated. Modulated differential scanning calorimetry (MDSC) was used to determine the glass transition temperature of both dry and hydrated PDLA samples. The absorption rate and total amounts of sorbed water by the polymer were determined by dynamic vapour sorption (DVS). Expansion behaviour of PDLA tablets was measured using thermal mechanical analysis (TMA). At 95% relative humidity all molecular weight grades of PDLA sorbed 1.1-1.3% w/w water, as was determined with DVS. MDSC showed glass transition temperature reductions of 10-11 degrees C for all molecular weight grades of PDLA in water. Volume expansion studies using TMA showed that the molecular relaxation time and equilibrium porosity of the tablets increased with molecular weight. The mean relaxation time increased exponentially with the temperature interval T(g)-T. The onset temperature of shape recovery of hydrated tablets was approximately 8 degrees C lower than for dry samples. Drug release was only slightly affected by molecular weight. It is concluded that volume expansion of compressed PDLA tablets is related to the glass transition behaviour, originates from water-induced and thermally stimulated shape memory behaviour and is therefore highly dependent on the molecular weight of PDLA.

Copolymers of 2-(dimethylamino)ethyl methacrylate with ethoxytriethylene glycol methacrylate or N-vinyl-pyrrolidone as gene transfer agents.

Random copolymers of 2-(dimethylamino)ethyl methacrylate (DMAEMA) with ethoxytriethylene glycol methacrylate (triEGMA) or N-vinylpyrrolidone (NVP) of different molecular weights and compositions were synthesized, characterized and evaluated as polymeric transfectants in vitro. All synthesized copolymers (comonomer fraction up to 66 mol%) were able to bind to DNA, yielding polymer-plasmid complexes (polyplexes). However, the polymer-plasmid ratio at which small complexes (size 0.2-0.3 microm) were formed, increased with increasing mole fraction of the comonomer. zeta-Potential measurements revealed that the polymer-plasmid ratio where charge neutralization of DNA occurred, increased with increasing mole fraction of triEGMA. The cytotoxicity of the copolymers, either complexed with DNA or in the free form, decreased with increasing mole fraction of both comonomers (triEGMA and NVP). This reduction was even more than what could be expected based on the DMAEMA mole fraction in the copolymer. The copolymers with a molecular weight up to 170¿ omitted¿000 had the same transfection capability as a homopolymer of comparable molecular weight. However, higher molecular weight copolymers showed a reduced transfection capability compared to the homopolymer, which was ascribed to the reduced capability to condense the size of plasmid. Transfection efficiency of polyplexes composed of copolymers with a low triEGMA content increased with increasing molecular weight. Although the copolymers with 50 mol% triEGMA were also better transfectants than the homopolymer, the transfection efficiency did not increase further with increasing molecular weight. Interestingly, NVP-DMAEMA copolymers synthesized by polymerization to high conversion showed both excellent DNA binding and condensing characteristics (polyplex size <0.3 microm) and transfection capabilities. This is ascribed to a synergistic effect of DMAEMA-rich copolymers and NVP-rich copolymers present in this system on the complex formation with plasmid DNA.