Rho-associated protein kinase (ROCK) inhibitors allow for causative glaucoma therapy. Unfortunately, topically applied ROCK inhibitors suffer from high incidence of hyperemia and low intraocular bioavailability. Therefore, we propose the use of poly (lactide-co-glycolide) (PLGA) microspheres as a depot formulation for intravitreal injection to supply outflow tissues with the ROCK inhibitor fasudil over a prolonged time. Fasudil-loaded microspheres were prepared by double emulsion solvent evaporation technique. The chemical integrity of released fasudil was confirmed by mass spectrometry. The biological activity was measured in cell-based assays using trabecular meshwork cells (TM cells), Schlemm’s canal cells (SC cells), fibroblasts and adult retinal pigment epithelium cells (ARPE-19). Cellular response to fasudil after its diffusion through vitreous humor was investigated by electric cell-substrate impedance sensing. Microspheres ranged in size from 3 to 67 µm. The release of fasudil from microspheres was controllable and sustained for up to 45 days. Released fasudil reduced actin stress fibers in TM cells, SC cells and fibroblasts. Decreased collagen gel contraction provoked by fasudil was detected in TM cells (~2.4-fold), SC cells (~1.4-fold) and fibroblasts (~1.3-fold). In addition, fasudil readily diffused through vitreous humor reaching its target compartment and eliciting effects on TM cells. No negative effects on ARPE-19 cells were observed. Since fasudil readily diffuses through the vitreous humor, we suggest that an intravitreal drug depot of ROCK inhibitors could significantly improve current glaucoma therapy particularly for patients with comorbid retinal diseases.
Water-free preparation of protein delivery systems has the potential to overcome the limitations of hydrogel depot systems such as off-target reactions, functional group hydrolysis, and limited loading capacity. However, a major roadblock in the development and use of these systems is administration as implantation is often required. In this study, we developed a biodegradable and water-free injectable protein delivery system via inverse electron demand Diels−Alder reaction between norbornene-and tetrazine-functionalized four-armed poly(ethylene glycol) macromonomers. 1:1 mixtures of these precursors gelled rapidly in situ, taking less than 11 s to reach their gelation point. Methyl substitution of tetrazine slowed the gelation time and increased the cross-linking density, whereas oxygen incorporation into norbornene changed the mechanical properties. Introduction of hydrolytically cleavable groups enabled biodegradability. Using phenyl carbamate and phenyl carbonate ester groups, we could tune the stability. Controlled release of the protein surrogate glucose oxidase was achieved over a period of 500 days. The novel preparation method presented here is a promising step toward the development of water-free injectable protein depots for controlled drug delivery.
Biopharmaceuticals are often lyophilized to improve their storage stability. Controlling ice nucleation during the freezing step of the lyophilization process is desired to increase homogeneity of product properties across a drug product batch and shorten the primary drying time. The present communication summarizes the process optimization of the freezing process when using vacuum-induced surface freezing to control ice nucleation, in particular for amorphous samples. We characterized freeze-dried samples for solid state properties, and compared these to uncontrolled nucleated samples using bovine serum albumin (BSA) as a model protein. Freezing parameters were optimized to obtain complete nucleation, adequate cake resistance during the subsequent lyophilization cycle, and elegant cakes. We highlight the challenges associated with vacuum-induced surface freezing and propose optimized freezing parameters to control ice nucleation, enabling manufacturing of amorphous samples.
Atherosclerosis is one of the most urgent global health
subjects,
causes millions of deaths worldwide, and is associated with enormous
healthcare costs. Macrophages are the root cause for inflammatory
onset and progression of the disease but are not addressed by conventional
therapy. Therefore, we used pioglitazone, which is a drug initially
used for diabetes therapies, but at the same time has great potential
regarding the mitigation of inflammation. As yet, this potential of
pioglitazone cannot be exploited, as drug concentrations at the target
site in vivo are not sufficient. To overcome this shortcoming, we
established PEG–PLA/PLGA-based nanoparticles loaded with pioglitazone
and tested them in vitro. Encapsulation of the drug was analyzed by
HPLC and revealed an outstanding encapsulation efficiency of 59% into
the nanoparticles, which were 85 nm in size and had a PDI of 0.17.
Further, uptake of our loaded nanoparticles in THP-1 macrophages was
comparable to the uptake of unloaded nanoparticles. On the mRNA level,
pioglitazone-loaded nanoparticles were superior to the free drug by
32% in increasing the expression of the targeted receptor PPAR-γ.
Thereby the inflammatory response in macrophages was ameliorated.
In this study, we take the first step toward an anti-inflammatory,
causal antiatherosclerotic therapy, using the potential of the already
established drug pioglitazone, and enable it to enrich at the target
site by using nanoparticles. An additional crucial feature of our
nanoparticle platform is the versatile modifiability of ligands and
ligand density, to achieve an optimal active targeting effect in the
future.
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