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.

A shift of paradigm: From avoiding nanoparticular complement activation in the field of nanomedicines to its exploitation in the context of vaccine development.

The complement system plays a central role in our innate immunity to fight pathogenic microorganisms, foreign and altered cells, or any modified molecule. Consequences of complement activation include cell lysis, release of histamines, and opsonization of foreign structures in preparation for phagocytosis. Because nanoparticles interact with the immune system in various ways and can massively activate the complement system due to their virus-mimetic size and foreign texture, detrimental side effects have been described after administration like pro-inflammatory responses, inflammation, mild to severe anaphylactic crisis and potentially complement activated-related pseudoallergy (CARPA). Therefore, application of nanotherapeutics has sometimes been observed with restraint, and avoiding or even suppressing complement activation has been of utmost priority. In contrast, in the field of vaccine development, particularly protein-based immunogens that are attached to the surface of nanoparticles, may profit from complement activation regarding breadth and potency of immune response. Improved transport to the regional lymph nodes, enhanced antigen uptake and presentation, as well as beneficial effects on immune cells like B-, T- and follicular dendritic cells may be exploited by strategic nanoparticle design aimed to activate the complement system. However, a shift of paradigm regarding complement activation by nanoparticular vaccines can only be achieved if these beneficial effects are accurately elicited and overshooting effects avoided.

Immunogenicity of a silica nanoparticle-based SARS-CoV-2 vaccine in mice.

Safe and effective vaccines have been regarded early on as critical in combating the COVID-19 pandemic. Among the deployed vaccine platforms, subunit vaccines have a particularly good safety profile but may suffer from a lower immunogenicity compared to mRNA based or viral vector vaccines. In fact, this phenomenon has also been observed for SARS-CoV-2 subunit vaccines comprising the receptor-binding domain (RBD) of the spike (S) protein. Therefore, RBD-based vaccines have to rely on additional measures to enhance the immune response. It is well accepted that displaying antigens on nanoparticles can improve the quantity and quality of vaccine-mediated both humoral and cell-mediated immune responses. Based on this, we hypothesized that SARS-CoV-2 RBD as immunogen would benefit from being presented to the immune system via silica nanoparticles (SiNPs). Herein we describe the preparation, in vitro characterization, antigenicity and in vivo immunogenicity of SiNPs decorated with properly oriented RBD in mice. We found our RBD-SiNP conjugates show narrow, homogeneous particle distribution with optimal size of about 100 nm for efficient transport to and into the lymph node. The colloidal stability and binding of the antigen was stable for at least 4 months at storage- and in vivo-temperatures. The antigenicity of the RBD was maintained upon binding to the SiNP surface, and the receptor-binding motif was readily accessible due to the spatial orientation of the RBD. The particles were efficiently taken up in vitro by antigen-presenting cells. In a mouse immunization study using an mRNA vaccine and spike protein as benchmarks, we found that the SiNP formulation was able to elicit a stronger RBD-specific humoral response compared to the soluble protein. For the adjuvanted RBD-SiNP we found strong S-specific multifunctional CD4+ T cell responses, a balanced T helper response, improved auto- and heterologous virus neutralization capacity, and increased serum avidity, suggesting increased affinity maturation. In summary, our results provide further evidence for the possibility of optimizing the cellular and humoral immune response through antigen presentation on SiNP.

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.

Multivalent display of engineered HIV-1 envelope trimers on silica nanoparticles for targeting and in vitro activation of germline VRC01 B cells.

Selective targeting of germline B cells with specifically designed germline-targeting HIV-1 envelope immunogens (GT-Env) is considered a feasible vaccination strategy to elicit broadly neutralizing antibodies (bnAbs). BnAbs are extremely valuable because they neutralize genetically distant viral strains at the same time. To overcome its inherently low affinity to germline B cells, the aim of the study was to present GT-Env via different immobilization strategies densely arrayed on the surface of nanoparticles. We engineered a prefusion-stabilized GT-Env trimer with affinity to VRC01 germline B cells using a bioinformatics-supported design approach. Distinct glycan modifications and amino acid substitutions yielded a GT-Env trimer which bound to the receptor with a KD of 11.5 µM. Silica nanoparticles with 200 nm diameter (SiNPs) were used for the multivalent display of the novel GT-Env with a 15 nm mean centre-to-centre spacing either by site-specific, covalent conjugation or at random, non-specific adsorption. Oriented, covalent GT-Env conjugation revealed better binding of structure dependent bnAbs as compared to non-specifically adsorbed GT-Env. In addition, GT-Env covalently attached activated a B cell line expressing the germline VRC01 receptor at an EC50 value in the nanomolar range (4 nM), while soluble GT-Env required 1,000-fold higher concentrations to induce signalling. The significantly lower GT-Env concentration was likely required due to avidity effects, which were in the picomolar range. Thus, low affinity antigens may particularly benefit from a particulate and multivalent delivery. In future, SiNPs are ideal to be modified in a modular design with various GT-Env variants that target different stages of germline and bnAb precursor B cells.

Distribution of Gold Nanoparticles in the Anterior Chamber of the Eye after Intracameral Injection for Glaucoma Therapy.

In glaucoma therapy, nanoparticles (NPs) are a favorable tool for delivering drugs to the outflow tissues of the anterior chamber of the eye where disease development and progression take place. In this context, a prerequisite is an efficient enrichment of NPs in the trabecular meshwork with minimal accumulation in off-target tissues such as the cornea, lens, iris and ciliary body. We evaluated the optimal size for targeting the trabecular meshwork by using gold NPs of 5, 60, 80 and 120 nm with a bare surface (AuNPs) or coated with hyaluronic acid (HA-AuNPs). NPs were compared regarding their colloidal stability, distribution in the anterior chamber of the eye ex vivo and cellular uptake in vitro. HA-AuNPs demonstrated an exceptional colloidal stability. Even after application into porcine eyes ex vivo, the HA coating prevented an aggregation of NPs inside the trabecular meshwork. NPs with a diameter of 120 nm exhibited the highest volume-based accumulation in the trabecular meshwork. Off-target tissues in the anterior chamber demonstrated an exceptionally low gold content. Our findings are particularly important for NPs with encapsulated anti-glaucoma drugs because a higher particle volume would be accompanied by a higher drug payload.

Augmenting the Immune Response against a Stabilized HIV-1 Clade C Envelope Trimer by Silica Nanoparticle Delivery.

The delivery of HIV-1 envelope (Env) trimer-based immunogens on the surface of nanoparticles holds promise to promote immunogenicity with the aim of inducing a potent, durable and broad neutralizing antibody (bnAb) response. Towards that goal, we examined the covalent conjugation of Env to 100 nm and 200 nm silica nanoparticles (SiNPs) to optimize conjugation density and attachment stability. Env was redesigned to enable site-specific cysteine-mediated covalent conjugation while maintaining its structural integrity and antigenicity. Env was anchored to different sized SiNPs with a calculated spacing of 15 nm between adjacent trimers. Both particle sizes exhibited high in vitro stability over a seven-day period. After attachment, 100 nm particles showed better colloidal stability compared to 200 nm particles. Importantly, the antigenic profile of Env was not impaired by surface attachment, indicating that the quaternary structure was maintained. In vitro Env uptake by dendritic cells was significantly enhanced when Env was delivered on the surface of nanoparticles compared to soluble Env. Furthermore, multivalent Env displayed efficiently activated B cells even at Env concentrations in the low nanomolar range. In mice, antibody responses to nanoparticle-coupled Env were stronger compared to the free protein and had equivalent effects at lower doses and without adjuvant.

Atherosclerosis: Conventional intake of cardiovascular drugs versus delivery using nanotechnology - A new chance for causative therapy?

Atherosclerosis is the leading cause of death in developed countries. The pathogenetic mechanism relies on a macrophage-based immune reaction to low density lipoprotein (LDL) deposition in blood vessels with dysfunctional endothelia. Thus, atherosclerosis is defined as a chronic inflammatory disease. A plethora of cardiovascular drugs have been developed and are on the market, but the major shortcoming of standard medications is that they do not address the root cause of the disease. Statins and thiazolidinediones that have recently been recognized to exert specific anti-atherosclerotic effects represent a potential breakthrough on the horizon. But their whole potential cannot be realized due to insufficient availability at the pathological site and severe off-target effects. The focus of this review will be to elaborate how both groups of drugs could immensely profit from nanoparticulate carriers. This delivery principle would allow for their accumulation in target macrophages and endothelial cells of the atherosclerotic plaque, increasing bioavailability where it is needed most. Based on the analyzed literature we conclude design criteria for the delivery of statins and thiazolidinediones with nanoparticles for anti-atherosclerotic therapy. Nanoparticles need to be below a diameter of 100 nm to accumulate in the atherosclerotic plaque and should be fabricated using biodegradable materials. Further, the thiazolidinediones or statins must be encapsulated into the particle core, because especially for thiazolidindiones the uptake into cells is prerequisite for their mechanism of action. For optimal uptake into targeted macrophages and endothelial cells, the ideal particle should present ligands on its surface which bind specifically to scavenger receptors. The impact of statins on the lectin-type oxidized LDL receptor 1 (LOX1) seems particularly promising because of its outstanding role in the inflammatory process. Using this pioneering concept, it will be possible to promote the impact of statins and thiazolidinediones on macrophages and endothelial cells and significantly enhance their anti-atherosclerotic therapeutic potential.

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.

Considerations for efficient surface functionalization of nanoparticles with a high molecular weight protein as targeting ligand.

Functionalization of nanoparticles with ligands is a powerful tool to achieve efficient targeting of receptors expressed on specific cell types. For optimal ligand-receptor interactions, the ligands should be attached on the nanoparticle surface in a predictable manner with specific orientations and density that preserve their bioactivity. While there are many publications on nanoparticles functionalized with small ligands that meet these requirements, achieving these conditions is particularly challenging for protein-based ligands of higher molecular weight. Proteins have complex and often fragile structures with numerous reactive residues, and they generally do not withstand harsh reaction conditions well. They are also prone to non-specific adsorption. Thus, conjugation strategies have to be considered carefully and optimized for each individual protein-based ligand as well as for the particle platform. In this study, we present a comprehensive approach for site-selective conjugation between aminated silica nanoparticles (SiNPs) and the single accessible thiol in human serum albumin (HSA) (66.5 kDa). We varied several reaction parameters including the density of amino groups on the particle surface, protein to amino group molar ratios, and linker length and evaluated their effect on colloidal stability, mode of protein attachment, protein density, and binding capacity of the tethered protein. We demonstrated that particle surface properties strongly impact covalent conjugation. For SiNPs with low amino group density (5,000 NH2/particle), only 25% of the available surface was covered with protein, and up to 90% of HSA was non-specifically adsorbed. Adjusting the molar ratio of HSA and lengthening the linker did not substantially increase the amount of covalently-attached ligand. In contrast, SiNPs with high amino group density (20,000 NH2/particle) showed high protein loading accompanied by low levels of non-specific adsorption. Using a short linker and 1:1 HSA to NH2 molar ratio resulted in 70% surface coverage with HSA molecules. The mode of attachment and protein density strongly impacted the functionality of the immobilized HSA. High non-specific adsorption resulted in the loss of its binding capacity, whereas predominately covalently-conjugated HSA showed binding affinities higher than that of soluble HSA and had a Kd value in the range of about 6 to 12 nM. Our findings indicate that reaction parameters should be carefully assessed to obtain site-selective and specifically oriented conjugation that maintains the protein's binding capacity. The approach presented here may serve as general instruction for the immobilization of high molecular weight targeting proteins to the surfaces of nanoparticles.

Silica particles incorporated into PLGA-based in situ-forming implants exploit the dual advantage of sustained release and particulate delivery.

Poly (lactic-co-glycolic acid) (PLGA) in situ-forming implants are well-established drug delivery systems for controlled drug release over weeks up to months. To prevent initial burst release, which is still a major issue associated with PLGA-based implants, drugs attached to particulate carriers have been encapsulated. Unfortunately, former studies only investigated the resulting release of the soluble drugs and hence missed the potential offered by particulate drug release. In this study, we developed a system capable of releasing functional drug-carrying particles over a prolonged time. First, we evaluated the feasibility of our approach by encapsulating silica particles of different sizes (500 nm and 1 µm) and surface properties (OH or NH2 groups) into in situ-forming PLGA implants. In this way, we achieved sustained release of particles over periods ranging from 30 to 70 days. OH-carrying particles were released much more quickly when compared to NH2-modified particles. We demonstrated that the underlying release mechanisms involve size-dependent diffusion and polymer-particle interactions. Second, particles that carried covalently-attached ovalbumin (OVA) on their surfaces were incorporated into the implant. We demonstrated that OVA was released in association with the particles as functional entities over a period of 30 days. The released particle-drug conjugates maintained their colloidal stability and were efficiently taken up by antigen presenting cells. This system consisting of particles incorporated into PLGA-based in situ-forming implants offers the dual advantage of sustained and particulate release of drugs as a functional unit and has potential for future use in many applications, particularly in single-dose vaccines.

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.

Critical design criteria for engineering a nanoparticulate HIV-1 vaccine.

Inducing a long-lasting as well as broad and potent immune response by generating broadly neutralizing antibodies is a major goal and at the same time the main challenge of preventive HIV-1 vaccine design. Immunization with soluble, stabilized and native-like envelope (Env) glycoprotein so far only led to low neutralization breadth and displayed low immunogenicity. A promising approach to generate a potent immune response is the presentation of Env on the surface of nanoparticles. In this review, we will focus on two key processes essential for the induction of immune response that can be addressed by specific features of nanoparticulate carriers: first, the trafficking to and within distinct compartments of the lymph node, and second, the use of multivalent Env display allowing for high avidity interactions. To optimize these pivotal steps critical design criteria should be considered for the presentation of Env on nanoparticles. These include an optimal particle size below 100 nm, distances between two adjacent Env antigens of approximately 10-15 nm, an appropriate orientation of Env, and finally, the stability of both the Env attachment and the nanoparticle platform. Hence, an interdisciplinary approach that combines a suitable delivery system and a straightforward presentation of the Env antigen may have the potential to drive the immune response towards increased breadth and potency.

Presentation of HIV-1 Envelope Trimers on the Surface of Silica Nanoparticles.

Inducing immune responses protecting from HIV infection or at least controlling replication poses a huge challenge to modern vaccinology. An increasingly discussed strategy to elicit a potent and broad neutralizing antibody response is the immobilization of HIV's trimeric envelope (Env) surface receptor on a nanoparticulate carrier. As a conceptual proof, we attached an Env variant (BG505 SOSIP.664) to highly stable and biocompatible silica nanoparticles (SiNPs) via site-specific covalent conjugation or nonspecific adsorption to SiNPs. First, we demonstrated the feasibility of SiNPs as platform for Env presentation by a thorough characterization process during which Env density, attachment stability, and antigenicity were evaluated for both formulations. Binding affinities to selected antibodies were in the low nanomolar range for both formulations confirming that the structural integrity of Env is retained after attachment. Second, we explored the recognition of SiNP conjugates by antigen presenting cells. Here, the uptake of Env attached to SiNPs via a site-specific covalent conjugation was 4.5-fold enhanced, whereas adsorbed Env resulted only in a moderate 1.4-fold increase compared with Env in its soluble form. Thus, we propose SiNPs with site-specifically and covalently conjugated Env preferably in a high density as a promising candidate for further investigations as vaccine platform.

Layer-by-Layer Assembled Nanoparticles for siRNA Delivery.

Nanoparticles synthesized via layer-by-layer processes are promising candidates for successful drug and gene delivery. Widespread use of the layer-by-layer technique has resulted from its accessibility to every lab; to generate nanoscale structures, layer-by-layer processes require common lab equipment of only modest quality and do not involve the use of organic solvents. In addition, a wide range of different starting materials can be flexibly combined, enabling the production of a nearly unlimited number of different nanoparticles (NP) with various physicochemical properties. Here we describe the manufacturing of poly(lactic-co-glycolic acid) NPs coated with siRNA for gene silencing. Positively charged polyethyleneimine and negatively charged nucleic acids form the polyelectrolyte shell. Finally, the NPs are functionalized with hyaluronic acid, a polysaccharide which targets the CD44 receptor.

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.

Intracameral Delivery of Layer-by-Layer Coated siRNA Nanoparticles for Glaucoma Therapy.

Glaucoma is the second leading cause of blindness worldwide, often associated with elevated intraocular pressure. Connective tissue growth factor (CTGF) is a mediator of pathological effects in the trabecular meshwork (TM) and Schlemm's canal (SC). A novel, causative therapeutic concept which involves the intracameral delivery of small interfering RNA against CTGF is proposed. Layer-by-layer coated nanoparticles of 200-260 nm with a final layer of hyaluronan (HA) are developed. The HA-coating should provide the nanoparticles sufficient mobility in the extracellular matrix and allow for binding to TM and SC cells via CD44. By screening primary TM and SC cells in vitro, in vivo, and ex vivo, the validity of the concept is confirmed. CD44 expression is elevated in glaucomatous versus healthy cells by about two- to sixfold. CD44 is significantly involved in the cellular uptake of HA-coated nanoparticles. Ex vivo organ culture of porcine, murine, and human eyes demonstrates up to threefold higher accumulation of HA compared to control nanoparticles and much better penetration into the target tissue. Gene silencing in primary human TM cells results in a significant reduction of CTGF expression. Thus, HA-coated nanoparticles combined with RNA interference may provide a potential strategy for glaucoma therapy.

Preventing Obstructions of Nanosized Drug Delivery Systems by the Extracellular Matrix.

Although nanosized drug delivery systems are promising tools for the treatment of severe diseases, the extracellular matrix (ECM) constitutes a major obstacle that endangers therapeutic success. Mobility of diffusing species is restricted not only by small pore size (down to as low as 3 nm) but also by electrostatic interactions with the network. This article evaluates commonly used in vitro models of ECM, analytical methods, and particle types with respect to their similarity to native conditions in the target tissue. In this cross-study evaluation, results from a wide variety of mobility studies are analyzed to discern general principles of particle-ECM interactions. For instance, cross-linked networks and a negative network charge are essential to reliably recapitulate key features of the native ECM. Commonly used ECM mimics comprised of one or two components can lead to mobility calculations which have low fidelity to in vivo results. In addition, analytical methods must be tailored to the properties of both the matrix and the diffusing species to deliver accurate results. Finally, nanoparticles must be sufficiently small to penetrate the matrix pores (ideally Rd/p < 0.5; d = particle diameter, p = pore size) and carry a neutral surface charge to avoid obstructions. Larger (Rd/p >> 1) or positively charged particles are trapped.

Hyaluronan as a promising excipient for ocular drug delivery.

Hyaluronan (HA) is a naturally occurring polysaccharide and well known for its exceptional properties such as high biocompatibility and biodegradability, along with a low immunogenicity. Besides its use for various biomedical applications it recently came into focus as a favorable excipient for the formulation of various ocular therapeutics. This review article summarizes the ocular distribution of HA and its most heavily investigated binding protein "cluster of differentiation 44" (CD44) which is the rationale for the clinical use of HA, primarily as an additive in ocular applications ranging from eye drops to contact lenses. Moreover, examples will be given for using HA in various pre-clinical approaches to generate entirely new therapeutics, most notably in the field of nanotechnology.