Prolonged delivery of HIV-1 vaccine nanoparticles from hydrogels.

Immunization is a straightforward concept but remains for some pathogens like HIV-1 a challenge. Thus, new approaches towards increasing the efficacy of vaccines are required to turn the tide. There is increasing evidence that antigen exposure over several days to weeks induces a much stronger and more sustained immune response compared to traditional bolus injection, which usually leads to antigen elimination from the body within a couple of days. Therefore, we developed a poly(ethylene) glycol (PEG) hydrogel platform to investigate the principal feasibility of a sustained release of antigens to mimic natural infection kinetics. Eight-and four-armed PEG macromonomers of different MWs (10, 20, and 40 kDa) were end-group functionalized to allow for hydrogel formation via covalent cross-linking. An HIV-1 envelope (Env) antigen in its trimeric (Envtri) or monomeric (Envmono) form was applied. The soluble Env antigen was compared to a formulation of Env attached to silica nanoparticles (Env-SiNPs). The latter are known to have a higher immunogenicity compared to their soluble counterparts. Hydrogels were tunable regarding the rheological behavior allowing for different degradation times and release timeframes of Env-SiNPs over two to up to 50 days. Affinity measurements of the VCR01 antibody which specifically recognizes the CD4 binding site of Env, revealed that neither the integrity nor the functionality of Envmono-SiNPs (Kd = 2.1 ± 0.9 nM) and Envtri-SiNPs (Kd = 1.5 ± 1.3 nM), respectively, were impaired after release from the hydrogel (Kd before release: 2.1 ± 0.1 and 7.8 ± 5.3 nM, respectively). Finally, soluble Env and Env-SiNPs which are two physico-chemically distinct compounds, were co-delivered and shown to be sequentially released from one hydrogel which could be beneficial in terms of heterologous immunization or single dose vaccination. In summary, this study presents a tunable, versatile applicable, and effective delivery platform that could improve vaccination effectiveness also for other infectious diseases than HIV-1.

Encapsulation of Pioglitazone into Polymer-Nanoparticles for Potential Treatment of Atherosclerotic Diseases.

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.

Injectable Anhydrous Poly(ethylene glycol) Polymer Liquids Form Protein Depot for Extended Controlled Release Applications Via Rapid In Situ Gelation.

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.

Angiopoietin-1 Mimetic Nanoparticles for Restoring the Function of Endothelial Cells as Potential Therapeutic for Glaucoma.

A root cause for the development and progression of primary open-angle glaucoma might be the loss of the Schlemm's canal (SC) cell function due to an impaired Angiopoietin-1 (Angpt-1)/Tie2 signaling. Current therapeutic options fail to restore the SC cell function. We propose Angpt-1 mimetic nanoparticles (NPs) that are intended to bind in a multivalent manner to the Tie2 receptor for successful receptor activation. To this end, an Angpt-1 mimetic peptide was coupled to a poly(ethylene glycol)-poly(lactic acid) (PEG-PLA) block co-polymer. The modified polymer allowed for the fabrication of Angpt-1 mimetic NPs with a narrow size distribution (polydispersity index < 0.2) and the size of the NPs ranging from about 120 nm (100% ligand density) to about 100 nm (5% ligand density). NP interaction with endothelial cells (HUVECs, EA.hy926) as surrogate for SC cells and fibroblasts as control was investigated by flow cytometry and confocal microscopy. The NP-cell interaction strongly depended on the ligand density and size of NPs. The cellular response to the NPs was investigated by a Ca2+ mobilization assay as well as by a real-time RT-PCR and Western blot analysis of endothelial nitric oxide synthase (eNOS). NPs with a ligand density of 25% opposed VEGF-induced Ca2+ influx in HUVECs significantly which could possibly increase cell relaxation and thus aqueous humor drainage, whereas the expression and synthesis of eNOS was not significantly altered. Therefore, we suggest Angpt-1 mimetic NPs as a first step towards a causative therapy to recover the loss of SC cell function during glaucoma.

Fasudil Loaded PLGA Microspheres as Potential Intravitreal Depot Formulation for Glaucoma Therapy.

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.

Causative glaucoma treatment: promising targets and delivery systems.

Glaucoma is one of the most common causes of blindness worldwide. Elevated intraocular pressure (IOP) is the major modifiable risk factor of the disease. Conventional therapy suffers from poor compliance, low bioavailability, and the lack of causative treatment options. To improve therapeutic success, it is crucial to identify major mediators of pathological changes associated with elevated IOP and to intervene at the molecular level. Here, we discuss relevant key functions of transforming growth factor-ß2 (TGF-ß2), connective tissue growth factor (CTGF), integrins, Rho-associated kinase (ROCK), and nitric oxide (NO) with regard to the onset of glaucoma, highlighting new drug delivery approaches for causative treatment.