Biodistribution of quantum dots in the kidney after intravenous injection.

The biodistribution of nanoparticles is a major subject of current nanomedical research. To date, however, the exact investigation of nanoparticle fate in the microenvironment of a main excretory organ, the kidney has largely been neglected. In this study, the biodistribution of polyethylene glycol-coated quantum dots (Qdots) with special focus on their interaction with the kidney is investigated. Upon intravenous injection, nanoparticles showed effective blood circulation in mice and significant renal accumulation after two hours. Histological analysis of the kidney revealed that Qdots were strongly associated to the intraglomerular mesangial cells. This preferential deposition of nanoparticles in the kidney mesangium is highly promising, since it could be of utmost value for site-specific treatment of severe kidney diseases like diabetic nephropathy in the future.

Ocular delivery systems for poorly soluble drugs: an in-vivo evaluation.

For highly potent but poorly water-soluble drugs like cyclosporine A, the development of aqueous formulations providing an increase of corneal drug tissue levels, and thus of bioavailability, to increase patient compliance is still a challenge. Therefore, we designed two water-based liquid application systems, an in-situ nanosuspension (INS) and a micellar solution (MS), and tested both formulations in vivo at the rabbit cornea for tolerability and the tissue uptake of CsA. The evaluation of the biological tolerability by periodical eye examination during 180 min and quantification in a defined grading system revealed that the INS evoked minimal to no irritations whereas the MS was perfectly tolerated. After the observation period, the rabbits were sacrificed and the corneal tissue levels of CsA were analyzed. The INS and the MS both showed high levels of 1683±430 ngCsA/gcornea and 826±163 ngCsA/gcornea, respectively, and exceeded drug tissue levels reported for Restasis(®) (350 ngCsA/gcornea) and cationic emulsions (750 ngCsA/gcornea). These results marked our INS and MS as outstanding novel approaches for the treatment of inflammatory corneal diseases.

The mechanism of protein release from triglyceride microspheres.

The purpose of this study was to reveal factors that have an impact on the protein release kinetics from triglyceride microspheres prepared by spray congealing. We investigated the effect of protein particle size, morphology and distribution on protein release from microspheres by confocal laser scanning microscopy (CLSM)(.) The microspheres were loaded with three types of model particles made of FITC-labeled bovine serum albumin: freeze dried protein, spherical particles obtained by precipitation in the presence of PEG and micronized material. Investigation by light microscopy and laser light diffraction revealed that the freeze dried material consisted mainly of app. 29 µm elongated shaped particles. The precipitated BSA consisted mainly of 9.0 µm diameter spherically shaped particles while the micronized protein prepared by jet milling consisted of 4.9 µm sized rounded particles of high uniformity. Microspheres were embedded into a cold-curing resin and cut with a microtome. Subsequent investigation by CLSM revealed major differences of distribution of the polydisperse protein particles inside the microsphere sections depending on the type of BSA that was used. Particles of micronized and precipitated protein were distributed almost throughout the microsphere cross section. The protein distribution had a marked impact on the release kinetics in phosphate buffer. Large protein particles led to a considerably faster release than small ones. By staining the release medium we demonstrated that in all three cases there was a strong correlation between protein release and buffer intrusion.

Synergistic effects of growth and differentiation factor-5 (GDF-5) and insulin on expanded chondrocytes in a 3-D environment.

OBJECTIVE: To investigate the effects of growth and differentiation factor-5 (GDF-5) alone or in combination with insulin on engineered cartilage from primary or expanded chondrocytes during 3-dimensional in vitro culture. DESIGN: Juvenile bovine chondrocytes were seeded either as primary or as expanded (passage 2) cells onto polyglycolic acid fiber meshes and cultured for 3 weeks in vitro. Additionally, adult human chondrocytes were grown in pellet culture after expansion (passage 2). The culture medium was supplemented either with GDF-5 in varying concentrations or insulin alone, or with combinations thereof. RESULTS: For primary chondrocytes, the combination of GDF-5 and insulin led to increased proliferation and construct weight, as compared to either factor alone, however, the production of glycosaminoglycans (GAG) and collagen per cell were not affected. With expanded bovine chondrocytes, the use of GDF-5 or insulin alone led to only very small constructs with no type II collagen detectable. However, the combination of GDF-5 (0.01 or 0.1 microg/ml) and insulin (2.5 microg/ml) yielded cartilaginous constructs and, in contrast to the primary cells, the observed redifferentiating effects were elicited on the cellular level independent of proliferation (increased production of GAG and collagen per cell, clear shift in collagen subtype expression with type II collagen observed throughout the construct). The synergistic redifferentiating effects of the GDF-5/insulin combination were confirmed with expanded adult human cells, also exhibiting a clear shift in collagen subtype expression on the mRNA and protein level. CONCLUSIONS: In combination with insulin, GDF-5 appears to enable the redifferentiation of expanded chondrocytes and the concurrent generation of cartilaginous constructs. The demonstration of these synergistic effects also for adult human chondrocytes supports the clinical relevance of the findings.

Injectable in situ forming depot systems: PEG-DAE as novel solvent for improved PLGA storage stability.

Injectable in situ forming depots (ISFD) that contain a peptide or a protein within a polymeric solution comprise an attractive, but challenging application system. Beyond chemical compatibility, local tolerability and acute toxicity, an important factor for an ISFD is its storage stability as a liquid. In this study, poly(D,L-lactide-co-glycolide) (PLGA) degradation in the presence of poly(ethyleneglycol) (PEG) as biocompatible solvent was investigated as a function of storage temperature and water content. The PLGA molecular weight (Mw) was determined by gel permeation chromatography (GPC), and monitored by NMR during degradation. Rapid PLGA degradation of 75% at 25 degrees C storage temperature was shown to be the result of a transesterification using conventional PEG as solvent. A significant improvement with only 3% Mw loss was obtained by capping the PEG hydroxy- with an alkyl- endgroup to have poly(ethyleneglycol) dialkylether (PEG-DAE). The formation of PEG-PLGA block co-polymers was confirmed by NMR, only for PEG300. Reaction rate constants were used to compare PLGA degradation dissolved in conventional and alkylated PEGs. The degradation kinetics in PEG-DAE were almost completely insensitive to 1% additional water in the solution. The transesterification of the hydroxy endgroups of PEG with PLGA was the major degradation mechanism, even under hydrous conditions. The use of PEG-DAE for injectable polymeric solutions, showed PLGA stability under the chosen conditions for at least 2 months. Based on the results obtained here, PEG-DAE appears to be a promising excipient for PLGA-based, parenteral ISFD.

Enhanced bone morphogenetic protein-2 performance on hydroxyapatite ceramic surfaces.

The immobilization of biomolecules on biomaterial surfaces allows for the control of their localization and retention. In numerous studies, proteins have been simply adsorbed to enhance the biological performance of various materials in vivo. We investigated the potential of surface modification techniques on hydroxyapatite (HA) ceramic discs in an in vitro approach. A novel method for protein immobilization was evaluated using the aminobisphosphonates pamidronate and alendronate, which are strong Ca chelating agents, and was compared with the established silanization technique. Lysozyme and bone morphogenetic protein-2 (BMP-2) were used to assess the suitability of the two surface modification methods with regard to the enzymatic activity of lysozyme and to the capacity of BMP-2 to stimulate the osteoblastic differentiation of C2C12 mouse myoblasts. After immobilization, a 2.5-fold increase in enzymatic activity of lysozyme was observed compared with the control. The alkaline phosphatase activity per cell stimulated by immobilized BMP-2 was 2.5-fold higher [9 x 10(-6) I.U.] than the growth factor on unmodified surfaces [2-4 x 10(-6) I.U.]. With regard to the increase in protein activity, both procedures lead to equivalent results. Thus, the bisphosphonate-based surface modification represents a safe and easy alternative for the attachment of proteins to HA surfaces.

Quantum dots - nano-sized probes for the exploration of cellular and intracellular targeting.

Nanoparticles emerged as promising tool in drug targeting, since, after appropriate modification, they are able to deliver their payload to specific sites, like tissues, cells, or even certain cellular organelles. In this context, the delivery of nanoparticles from the circulation into the target cells represents a crucial step. Here, model drug delivery systems such as quantum dots are ideal candidates to elucidate this process in more detail, since they provide outstanding features like a small and uniform size, unique optical properties for most sensitive detection and modifiable surfaces. Recent progress in the surface chemistry of quantum dots expanded their use in biological applications, reduced their cytotoxicity and rendered quantum dots a powerful tool for the investigation of distinct cellular processes, like uptake, receptor trafficking and intracellular delivery. In this review, we will not only describe the ideal attributes of QDs for biological applications and imaging but also their distinct specific and non-specific pathways into the cells as well as their intracellular fate.

Polymers and nanoparticles: intelligent tools for intracellular targeting?

In recent years, a new generation of drugs has entered the pharmaceutical market. Some are more potent, but some are also more toxic and thus, therapeutical efficacy may be hindered, and severe side effects may be observed, unless they are delivered to their assigned place of effect. Those targets are not only certain cell types, moreover, in cancer therapy for example, some drugs even have to be targeted to a specific cell organelle. Those targets in eukaryotic cells include among others endo- and lysosomes, mitochondria, the so-called power plants of the cells, and the biggest compartment with almost all the genetic information, the nucleus. In this review, we describe how the drugs can be directed to specific subcellular organelles and focus especially on synthetic polymers and nanoparticles as their carriers. Furthermore, we portray the progress that has been accomplished in recent years in the field of designing the carriers for efficient delivery into these target structures. Yet, we do not fail to mention the obstacles that still exist and are preventing polymeric and nanoparticular drug carrier systems from their broad application in humans.

Methoxy poly(ethylene glycol)--low molecular weight linear polyethylenimine-derived copolymers enable polyplex shielding.

Targeted gene delivery relies on the development of materials that allow for the formation of small neutrally charged particles of sufficient colloidal stability preventing non-specific interactions with cells. In order to identify a copolymer composition that combines adequate plasmid DNA (pDNA) compaction with an efficient charge-shielding effect, we synthesized a series of copolymers by covalent linkage of activated 5 or 20 kDa linear methoxy poly(ethylene glycol) (mPEG) or 10 kDa two-arm-mPEG to non-toxic low molecular weight (2.6 and 4.6 kDa) linear polyethylenimine (lPEI) at different molar ratios (mPEG-lPEI copolymers). All of the copolymers condensed pEGFP-N1 pDNA to form nanoparticles with hydrodynamic diameters between 150 and 420 nm - sizes that were maintained for the entire duration of measurement. PEGylated complexes exhibited a reduced particle stability in comparison to the unmodified lPEI-pDNA polyplexes, determined by gel retardation assays and DNase I experiments. Copolymer-pDNA complexes exhibited a zeta potential between -4 and 6 mV, strongly depending on the dispersion medium applied (0.15M NaCl or 5% glucose supplemented with serum-free cell culture medium). The transfection efficacy, determined in CHO-K1 (between 0.28+/-0.08% and 1.92+/-0.46%) and HeLa (between 1.02+/-0.19% and 3.53+/-0.30%) cells, was significantly reduced compared to lPEI-pDNA particles (between 3.2+/-1.3% and 38.8+/-5.5%). The architecture of the copolymer, the molecular weight of the lPEI residue, and the supplementation of endosomolytic agents (saccharose, chloroquine) all failed to impact the efficacy of gene transfer. Uptake studies, based on Confocal Laser Scanning Microscopy (CLSM) imaging and flow cytometry analysis, suggest that the use of mPEG5/3-lPEI2.6, mPEG10/2-lPEI2.6, and mPEG20-lPEI4.6 lowers unspecific internalization of the corresponding transfection complexes. This provides an ideal basis for the development of transfection vehicles for targeted gene transfer.

Towards controlled release of BDNF--manufacturing strategies for protein-loaded lipid implants and biocompatibility evaluation in the brain.

It was the aim of this study to establish triglyceride matrices as potential carriers for long-term release of brain-derived neurotrophic factor (BDNF), a potential therapeutic for Huntington's disease. First, four different manufacturing strategies were investigated with lysozyme as a model substance: either lyophilized protein was mixed with lipid powder, or suspended in organic solution thereof (s/o). Or else, an aqueous protein solution was dispersed by w/o emulsion in organic lipid solution. Alternatively, a PEG co-lyophilization was performed prior to dispersing solid protein microparticles in organic lipid solution. After removal of the solvent(s), the resulting powder formulations were compressed at 250 N to form mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight. Protein integrity after formulation and release was evaluated from an enzyme activity assay and SDS-PAGE. Confocal microscopy revealed that the resulting distribution of FITC-lysozyme within the matrices depended strongly on the manufacturing method, which had an important impact on matrix performance: matrices with a very fine and homogeneous protein distribution (PEG co-lyophilization) continually released protein for 2 months. The other methods did not guarantee a homogeneous distribution and either failed in sustaining release for more than 1 week (powder mixture), completely liberating the loading (s/o dispersion) or preserving protein activity during manufacturing (w/o emulsion, formation of aggregates and 25% activity loss). Based on these results, miniature-sized implants of 1 mm diameter, 0.8 mm height and 1 mg weight were successfully loaded by the PEG co-lyophilization method with 2% BDNF and 2% PEG. Release studies in phosphate buffer pH 7.4 at 4 and 37 degrees C revealed a controlled release of either 20 or 60% intact protein over one month as determined by ELISA. SDS-PAGE detected only minor aggregates in the matrix during release at higher temperature. In vivo evaluation of lipid cylinders in the striatum of rat brains revealed a biocompatibility comparable to silicone reference cylinders.

Influence of wettability and surface activity on release behavior of hydrophilic substances from lipid matrices.

The aim of this study was to investigate the role of matrix and drug properties on controlled release from triglyceride matrices. Mini-cylinders of 2 mm diameter, 2.2 mm height and 7 mg weight were produced by compression of lipid powder obtained by using a polyethylene glycol (PEG) co-lyophilization method for the model substances lysozyme and FITC-dextran (Mw 4000 Da). Lysozyme was released with decreasing velocity from glyceryl trilaurate, -myristate, -palmitate and -stearate for more than 14 months. Release correlated well with triglyceride lipophilicity defined by the chain length of the fatty acids. Contact angle measurements and the analysis of buffer penetration visualized by confocal microscopy emphasized the role of matrix wettability as a prerequisite for release. A comparison with FITC-dextran revealed that the protein itself enhances matrix wettability and hence its release due to its surface active properties. FITC-dextran remained trapped within the matrix only to be released at lower compression force or after the addition of surfactant. Protein added externally to the release buffer at 0.1% (w/v) was not efficient in lowering the contact angle and increasing the release rate of FITC-dextran. Tween 20 and 81 could be used in different concentrations (0.1, 0.01 and 0.001% (w/v)) to alter lysozyme and FITC-dextran release profiles: resulting release rates showed a close dependence on the contact angle of the respective release medium and triglyceride matrix material. However, both Tweens seem to act not only by reducing the release medium contact angle but also by moderately affecting interparticulate adhesion of the matrix material.

Programmable implants--from pulsatile to controlled release.

The aim of this study was to develop programmable implants with a reproducible delayed onset of release followed by several weeks of controlled release. For this purpose, a drug-loaded core was embedded into a drug-free bulk-eroding poly(D,L lactic-co-glycolic acid) or poly(D,L lactic acid) mantle. The manufacturing procedure was established and optimized for three mantle materials, which showed delay times ranging from 7 to 83 days. Triglycerides with fatty acid chain lengths from C12 to C18 were investigated as core materials, producing release periods from 2 to 16 weeks. Concomitantly, applying a convolution/deconvolution model showed the possibility of theoretical prediction of the resulting release profiles.

Biocompatibility and erosion behavior of implants made of triglycerides and blends with cholesterol and phospholipids.

Triglycerides are a promising class of material for the parenteral delivery of drugs and have become the focus of tremendous research efforts in recent years. The aim of this study was to investigate the biocompatibility of glyceroltripalmitate as well as the influence of cholesterol and distearoyl-phosphatidyl-choline (DSPC) on the erosion behavior of the lipid. For these investigations, two in vivo studies were carried out, in which cylindrical matrices of 2 mm diameter were manufactured and subcutaneously implanted in immunocompetent NMRI-mice. After excision of the implants, tissue reactions of the animals as well as changes in the weight, shape and microstructure of the implants were investigated. The triglyceride and cholesterol showed good biocompatibility, as indicated by their minimal encapsulation in connective tissue and the absence of inflammatory reactions. Increasing the levels of phospholipid in the implants, however, led to an increased inflammatory reaction. In contrast to cholesterol, which did not affect erosion, the incorporation of DSPC into the triglyceride matrices led to clearly visible signs of degradation.

In vitro investigation of lipid implants as a controlled release system for interleukin-18.

Operating on the inductive and effective phases of an anti-tumor immune response and uncovering pivotal functions that may reduce cancer cell growth, interleukin-18 (IL-18) appears to be an attractive candidate for the sustained local adjuvant immunotherapeutic treatment of brain gliomas. The objective of this work was to develop IL-18 loaded lipid implants as a controlled delivery system. For the preparation of protein loaded triglyceride matrix material, a solid-in-oil (s/o) dispersion technique was chosen for which protein particles in the micrometer range were first prepared by co-lyophilization with polyethylene glycol (PEG). Implants of 1 mm diameter, 1.8 mm height and 1.8 mg weight were manufactured by compression of the powder mixture in a specially designed powder compacting tool. The in vitro release behavior of 125I-Bolton-Hunter-radiolabeled IL-18 was assessed in a continuous-flow system. A cell culture assay was established for the determination of bioactivity of released IL-18. Implants showed a continuous release of 10-100 ng IL-18 per day for 12 days. A progressive integrity loss was observed with ongoing release, which would be related to protein degradation during incubation. The initially released fraction proved complete retention of bioactivity, indicating that the manufacturing procedure had no detrimental effects on protein stability.

Drug release from lipid-based implants: elucidation of the underlying mass transport mechanisms.

The aim of this study was to better understand the mass transport mechanisms involved in the control of drug release from lipid-based implants. Different types of triglyceride-based cylinders were prepared by compression. Glycerol-trilaurate, -trimyristate, -tripalmitate and -tristearate were used as model lipids, lysozyme and pyranine as model drugs. The effects of several formulation and processing parameters on the resulting drug release kinetics in phosphate buffer pH 7.4 were studied and the obtained results analyzed using Fick's second law of diffusion. Interestingly, lysozyme release from implants prepared by compression of a lyophilized emulsion (containing dissolved drug and lipid) was found to be purely diffusion-controlled, irrespective of the type of triglyceride. In contrast, the dominating release mechanism depended on the type of lipid in the case of pyranine-loaded implants prepared by compression of a lyophilized lipid-drug solution: with glycerol-trilaurate and -tristearate the systems were found to be purely diffusion-controlled, whereas also other mass transport phenomena are of importance in glycerol-trimyristate and -tripalmitate-based devices. Similarly, changes in the size of the compressed lipid-drug particles, drug loading and compression force significantly affected the underlying release mechanisms. The addition of a drug-free, poly(lactic-co-glycolic acid) (PLGA)-based coating around the implants delayed the onset of pyranine release for about 20 days. Interestingly, the subsequent drug release was purely diffusion-controlled, irrespective of the type of triglyceride. Also the addition of different amounts (and particle size fractions) of saccharose to pyranine-loaded implants led to purely diffusion-controlled drug release.

Paclitaxel-loaded microparticles and implants for the treatment of brain cancer: preparation and physicochemical characterization.

The aim of this study was to prepare different types of paclitaxel-loaded, PLGA-based microparticles and lipidic implants, which can directly be injected into the brain tissue. Releasing the drug in a time-controlled manner over several weeks, these systems are intended to optimize the treatment of brain tumors. The latter is particularly difficult because of the blood-brain barrier (BBB), hindering most drugs to reach the target tissue upon systemic administration. Especially paclitaxel (being effective for the treatment of ovarian, breast, lung and other cancers) is not able to cross the BBB to a notable extent since it is a substrate of the efflux transporter P-glycoprotein. Both, biodegradable microparticles as well as small, cylindrical, glycerol tripalmitate-based implants (which can be injected using standard needles) were prepared with different paclitaxel loadings. The effects of several formulation and processing parameters on the resulting drug release kinetics were investigated in phosphate buffer pH 7.4 as well as in a diethylnicotinamide (DENA)/phosphate buffer mixture. Using DSC, SEM, SEC and optical microscopy deeper insight into the underlying drug release mechanisms could be gained. The presence of DENA in the release medium significantly increased the solubility of paclitaxel, accelerated PLGA degradation, increased the mobility of the polymer and drug molecules and fundamentally altered the geometry of the systems, resulting in increased paclitaxel release rates.

Bone morphogenetic protein 9: a potent modulator of cartilage development in vitro.

Few publications describe the activity of bone morphogenetic protein-9 (BMP-9), but the consensus of these largely in vivo studies is that while BMP-9 can induce ectopic bone formation at relatively large concentrations, it is primarily active in non-skeletal locations--including the liver, nervous system and marrow. To study the effects of BMP-9 on chondrogenesis in a well-defined environment, calf articular chondrocytes were seeded onto biodegradable PGA scaffolds. The resulting cell-polymer constructs were cultured in either control medium or medium supplemented with 1, 10, 50 or 100 ng/ml of BMP-9. After 4 weeks of in vitro culture, all concentrations of BMP-9 increased the total mass of the constructs, and the amounts of collagen, glycosaminoglycans (GAG) and cells per construct. On a mass percentage basis, BMP-9 tended to increase GAG, to decrease the relative amount of collagen and had little effect on the relative amount of cells. BMP-9 elicited qualitatively similar responses as BMP-2, -12 and -13. However, in contrast to BMP-12 and -13, BMP-9 (at concentrations > or = 10 ng/ml) induced hypertrophic chondrocyte formation and was the only BMP tested to induce mineralization. Taken together, these data suggest that BMP-9 is a potent modulator of cartilage development in vitro.