ABSTRACT
Dickkopf-1 (DKK1), a Wnt inhibitor secreted by bone marrow stromal cells (MSC), is known to play an important role in long-term non-union bone fracture defects and glucocorticoid induced osteoporosis. Mitigating its effects in early bone defects could improve osteogenesis and bone defect healing. Here, we applied a biomaterial strategy to deplete a defect environment from DKK1 by scavenging the protein via a macromer-based biomaterial covalently decorated with sulfated hyaluronan (sHA3). The material consisted of cross-copolymerized three-armed macromers with a small anchor molecule. Using the glycidyl anchor, polyetheramine (ED900) could be grafted to the material to which sHA3 was efficiently coupled in a separate step. For thorough investigation of material modification, flat material surfaces were generated by fabricating them on glass discs. The binding capability of sHA3 for DKK1 was demonstrated in this study by surface plasmon resonance measurements. Furthermore, the surfaces demonstrated the ability to scavenge and inactivate pathologic amounts of DKK1 from complex media. In a combinatory approach with Wnt3a, we were able to demonstrate that DKK1 is the preferred binding partner of our sHA3-functionalized surfaces. We validated our findings in a complex in vitro setting of differentiating SaOS-2 cells and primary hMSC. Here, endogenous DKK-1 was scavenged resulting in increased osteogenic differentiation indicating that this is a consistent biological effect irrespective of the model system used. Our study provides insights in the mechanisms and efficiency of sHA3 surface functionalization for DKK1 scavenging, which may be used in a clinical context in the future.
Subject(s)
Biocompatible Materials , Osteogenesis , Cell Differentiation , Hyaluronic Acid , SulfatesABSTRACT
The linear biodegradable polyester poly(glycerol adipate) (PGA) was synthesized via enzymatic polycondensation using lipase B from Candida antarctica (CAL-B). Every monomer unit of PGA possesses a pendant hydroxyl group which is responsible for the hydrophilic character and moisture swelling. These OH groups were esterified to different degrees with the anti-inflammatory drug indomethacin in order to create a prodrug with a pH-sensitive linker for modified drug release. The structure of the conjugates was determined via ATR FT-IR spectroscopy, NMR spectroscopy, GPC and UV/VIS spectroscopy. The physical properties of polymers with different drug load were investigated using DSC, contact angle measurements and oscillatory rheology. Drug release was monitored over one month in vitro. A very slow, but continuous release was observed in PBS. Slightly acidic conditions and lipase activity are accelerating the indomethacin release. Therefore, poly(glycerol adipate) - indomethacin conjugates are promising prodrugs for the local sustained release of indomethacin.
Subject(s)
Drug Compounding , Indomethacin/chemistry , Polyesters/chemistry , Delayed-Action Preparations , Spectroscopy, Fourier Transform InfraredABSTRACT
RNA interference (RNAi) is a promising technique to treat severe diseases on a pre-protein level. We and others postulate that the release of nanoparticle-complexed small interfering RNA (siRNA) from implanted biomaterials could provide structural support for tissue repair, combined with local siRNA transfection of invading and regenerating cells. In this study, we systematically investigated cross-linked gelatin based hydrogel formulations (cGEL) as degradable controlled release matrices for siRNA. Aiming at the definition of correlations between cGEL composition, siRNA nanoparticle formulation, release kinetics of complexed siRNA and transfection efficiency, we combined five different cGEL formulations and three transfection systems, i.e. polyplexes with polyethyleneimine (PEI), PEI in combination with liposomes (lipopolyplexes) and polyplexes based on tyrosin-modified PEI (P10Y). It was found that the distribution of these poly-/lipopolyplexes, when applied onto the negatively charged hydrogels, was strongly dependent on their zeta potential. Furthermore, siRNA release from the hydrogel was a multifactorial process, as diffusion, hydrogel degradation and nanoparticle decomplexation overlapped over time. This resulted in a prolonged release of siRNA for up to 21days. In the case of PEI complexes and lipopolyplexes, release kinetics depended on the cGEL formulation. In contrast, when employing P10Y polyplexes, an initial burst release was observed with no further release thereafter. Silencing activity was determined using constitutively luciferase-expressing SKOV-3-Luc reporter cells. Surface and bulk porosity in hydrogels was introduced by addition of soluble polyethylene glycol during fabrication, leading to improved knockdown. The rapid onset of knockdown efficacy will also provide the basis for the determination of long-term effects.
Subject(s)
Gelatin/chemistry , Hydrogels/chemistry , RNA, Small Interfering/administration & dosage , Transfection/methods , Cell Line, Tumor , Humans , Polyethyleneimine , RNA InterferenceABSTRACT
Toward a new generation of improved nerve guidance conduits (NGCs), novel biomaterials are required to address pressing clinical shortcomings in peripheral nerve regeneration (PNR) and to promote biological performance. A dual-component hydrogel system formed by cross-linking reaction between maleic anhydride groups in an oligomeric building block for cross-linking of free amine functionalities in partially hydrolyzed collagen is formulated for continuous processing and NGC fabrication. The influence of the gelation base is optimized for processing from a double syringe delivery system with a static mixer. A hydrophilic low-concentrated base was introduced to control network formation and to utilize highly reactive macromers for gelation. Cross-linking extent and building block conversion were improved and homogenous monoliths were fabricated. Chemically derivatized hydrogels were obtained by conversion of a fraction of anhydride groups in the oligomeric precursor with monovalent primary amine-containing grafting molecules prior to gelation. Network stability in functionalized hydrogels was maintained and cationic moieties were implement to the gel that promoted in vitro cell attachment and spreading irrespective of mechanical stiffness. A molding strategy was introduced that allowed for fabrication of flexible tubular conduits in tunable dimensions and with chemically patterned structures. These hydrogel-based conduits hold promise for the next generation NGCs with integrated chemical cues for PNR.
Subject(s)
Biocompatible Materials/chemistry , Biocompatible Materials/pharmacology , Hydrogels/chemistry , Hydrogels/pharmacology , Nerve Regeneration/drug effects , Tissue Scaffolds , Peptides/chemistryABSTRACT
The function of a photocrosslinked poly(propylene fumarate) (PPF)/poly(N-vinyl pyrrolidone) (PVP) matrix for the sustained release of three ophthalmic model drugs, acetazolamide (AZ), dichlorphenamide (DP), and timolol maleate (TM), was investigated. The drugs differ in molecular weight and degree of dissociation in aqueous environments; both are parameters that significantly influence drug diffusivity. AZ, DP, and TM-loaded cylindrical rods (10 mm length, 0.6 mm diameter) were fabricated by photoinduced cross-copolymerization of PPF and N-vinyl pyrrolidone (NVP) in molds. The released amounts of AZ, DP, TM, and NVP were determined by high-performance liquid chromatography (HPLC). The effects of drug properties and loading on the release kinetics were investigated. The in vitro release of AZ, DP, and TM was well sustained from the polymer matrices over a period of approximately 210, 270, and 250 days, respectively. The release kinetics correlated with the HPLC retention profiles of the different drugs. Following a small initial burst release (<10%), a dual modality release controlled by diffusion and bulk erosion was found for all drugs. Drug release rates of up to 4 microg/day were reached. Matrix drug loading generally affected the extent of the burst release, release kinetics, as well as the matrix water content and matrix degradation that were determined gravimetrically. Microcomputed tomography was used to image structural and dimensional changes of the devices. A preliminary rabbit implantation study revealed promising ocular biocompatibility of drug-free PPF/PVP matrices. All results indicate the potential of photocrosslinked PPF-based matrices as polymeric carriers for long-term ophthalmic drug delivery.
Subject(s)
Biocompatible Materials/chemistry , Drug Carriers/chemistry , Drug Delivery Systems , Eye/metabolism , Fumarates/metabolism , Polypropylenes/metabolism , Acetazolamide/administration & dosage , Acetazolamide/chemistry , Animals , Antihypertensive Agents/administration & dosage , Antihypertensive Agents/chemistry , Carbonic Anhydrase Inhibitors/administration & dosage , Carbonic Anhydrase Inhibitors/chemistry , Chromatography, High Pressure Liquid , Dichlorphenamide/administration & dosage , Dichlorphenamide/chemistry , Diuretics/administration & dosage , Diuretics/chemistry , Eye/anatomy & histology , Fumarates/chemistry , Implants, Experimental , Molecular Structure , Polypropylenes/chemistry , Rabbits , Timolol/administration & dosage , Timolol/chemistryABSTRACT
In this work, different modifications of photo-cross-linked poly(propylene fumarate)/poly(N-vinyl pyrrolidone) (PPF/PNVP) matrices were studied for their effect on the release kinetics of two ophthalmic drugs. The hydrophilicity of solid PPF/PNVP matrices loaded with acetazolamide (AZ) or timolol maleate (TM) was increased by adding various amounts of poly(ethylene glycol) (PEG) or by increasing the amount of N-vinyl pyrrolidone (NVP) in the polymer mixture prior to cross-linking. The in vitro release studies that utilized high-performance liquid chromatography for quantification revealed highly accelerated drug release from the matrices with increasing contents of the hydrophilic modifier. AZ was released from matrices containing 5% PEG in 56 days, which equals approximately 25% of the release period found for the unmodified matrices. A comparable acceleration in drug release was found for TM-loaded samples modified with 5% PEG. These studies further revealed that 1% PEG is sufficient to shorten the TM release duration by one-third. A significant acceleration in drug release was also found for the samples that were fabricated from a PPF-NVP mixture with increased NVP content. Matrix water content and erosion were assessed gravimetrically. Micro-computed tomography was used to image structural changes of the release systems and shed light on the drug-release mechanism. This study showed that hydrophilic matrix modifications of PPF/PNVP matrices accelerate the drug release of two ophthalmic drugs and represent a suitable tool to adjust drug-release rates from PPF-based matrices for different therapeutic needs.
Subject(s)
Drug Carriers/chemistry , Drug Delivery Systems , Fumarates/chemistry , Ophthalmic Solutions/chemistry , Polymers/chemistry , Polypropylenes/chemistry , Acetazolamide/chemistry , Acetazolamide/metabolism , Antihypertensive Agents/chemistry , Antihypertensive Agents/metabolism , Diuretics/chemistry , Diuretics/metabolism , Drug Administration Routes , Eye/chemistry , Eye/metabolism , Fumarates/chemical synthesis , Molecular Structure , Ophthalmic Solutions/metabolism , Polyethylene Glycols/chemistry , Polypropylenes/chemical synthesis , Timolol/chemistry , Timolol/metabolismABSTRACT
The objective of this study was to investigate the effect of poly(propylene fumarate) (PPF) molecular weight on the release kinetics of two ophthalmic model drugs, acetazolamide (AZ) and timolol maleate (TM), from matrices prepared by photo-induced copolymerization of PPF with N-vinyl pyrrolidone (NVP). PPF macromers of different number average molecular weight (M(n)) and polydispersity index (PI) were used in the experiments. Photo-crosslinked matrices were loaded with 5wt.% AZ or TM. The amounts of released drug and NVP were determined using high-performance liquid chromatography (HPLC). The release kinetics of both drugs was influenced by the molecular weight of the constituent PPF macromer. An increased M(n) led to an increased burst release and an accelerated drug release. Dependent on the PPF M(n), the initial AZ loading was released within periods ranging from 35 days (M(n) = 3670, PI = 1.9) to 220 days (M(n) = 2050, PI=1.5). TM-loaded matrices revealed similar release kinetics dependent on the PPF M(n). The amount of released NVP from photo-crosslinked matrices during the course of a release experiment was independent of the PPF M(n) for both drugs. Matrix swelling and erosion were determined gravimetrically. The network structures of non-loaded matrices were further characterized by determining their crosslinking densities and the relative double bond conversions of fumaric acid (FAA) and NVP. Independent of PPF M(n), PPF and NVP similarly participated in the formation of the PPF/polyNVP copolymer network. The observed differences in drug release might therefore be explained by differences in the microstructural organization of the copolymer networks. Overall, the results demonstrate that drug release kinetics from photo-crosslinked PPF/polyNVP matrices is strongly dependent on the M(n) of the PPF macromer.
Subject(s)
Acetazolamide/administration & dosage , Biocompatible Materials/chemistry , Drug Carriers/chemistry , Fumarates/chemistry , Polypropylenes/chemistry , Timolol/administration & dosage , Fumarates/radiation effects , Kinetics , Molecular Weight , Photochemistry , Polypropylenes/radiation effects , Pyrrolidinones/chemistry , Pyrrolidinones/metabolismABSTRACT
This study sought to develop an injectable formulation for long-term ocular delivery of fluocinolone acetonide (FA) by dissolving the anti-inflammatory drug and the biodegradable polymer poly(propylene fumarate) (PPF) in the biocompatible, water-miscible, organic solvent N-methyl-2-pyrrolidone (NMP). Upon injection of the solution into an aqueous environment, a FA-loaded PPF matrix is precipitated in situ through the diffusion/extraction of NMP into surrounding aqueous fluids. Fabrication of the matrices and in vitro release studies were performed in phosphate buffered saline at 37 degrees C. Drug loadings up to 5% were achieved. High performance liquid chromatography was employed to determine the released amount of FA. The effects of drug loading, PPF content of the injectable formulation, and additional photo-crosslinking of the matrix surface were investigated. Overall, FA release was sustained in vitro over up to 400 days. After an initial burst release of 22 to 68% of initial FA loading, controlled drug release driven by diffusion and bulk erosion was observed. Drug release rates in a therapeutic range were demonstrated. Release kinetics were found to be dependent on drug loading, formulation PPF content, and extent of surface crosslinking. The results suggest that injectable, in situ formed PPF matrices are promising candidates for the formulation of long-term, controlled delivery devices for intraocular drug delivery.
Subject(s)
Eye , Fluocinolone Acetonide/analogs & derivatives , Fumarates/chemistry , Glucocorticoids/chemistry , Polypropylenes/chemistry , Delayed-Action Preparations/chemistry , Fluocinolone Acetonide/chemistry , Kinetics , Pyrrolidinones/chemistryABSTRACT
Intraocular drug delivery systems made from biodegradable polymers hold great potential to effectively treat chronic diseases of the posterior segment of the eye. This study is based on the hypothesis that crosslinked poly(propylene fumarate) (PPF)-based matrices are suitable long-term delivery devices for the sustained release of the anti-inflammatory drug fluocinolone acetonide (FA) due to their hydrophobicity and network density. FA-loaded rods of 10 mm length and 0.6 mm diameter were fabricated by photo-crosslinking PPF with N-vinyl pyrrolidone (NVP). The released amounts of FA and NVP were determined by HPLC analysis. The effects of drug loading and the ratio of PPF to NVP on the release kinetics were investigated using a 2(3-1) factorial design. Overall, FA release was sustained in vitro over almost 400 days by all tested formulations. Low burst release was followed by a dual modality release controlled by diffusion and bulk erosion with release rates up to 1.7 microg/day. The extent of the burst effect and the release kinetics were controlled by the drug loading and the matrix composition. Matrix water content and degradation were determined gravimetrically. Micro-computed tomography was used to image structural and dimensional changes of the devices. The results show that photo-crosslinked PPF-based matrices are promising long-term delivery devices for intraocular drug delivery.