Polyethylenimine-based nucleic acid delivery

With recent advances in molecular biology and the sequencing of the human genome, nucleic acids are expected to engage a pivotal position in the development of novel therapeutic concepts. Although viral vectors are very effective and are capable of many of the required tasks for efficient gene transfer, their broad use is limited by severe safety risks [1,2]. Therefore, this work focused on the use of polymers as non-viral vehicles for nucleic acid delivery. Among various polymers, polyethylenimine (PEI) has become a gold standard for nucleic acid delivery [3]. The relatively high transfection efficiency of branched PEI (BPEI) -based delivery systems is seriously afflicted by the cytotoxicity of the polymer. In contrast, the linear form (LPEI) has a particularly high gene expression accompanied by a lower cytotoxicity, but nevertheless the transfection efficiency of LPEI 22 kDa (ExGen®) cannot be enhanced beyond a certain limit due to cytotoxicity. Therefore, the ultimate goal of this thesis was to identify strategies towards effective PEI-based delivery systems with low cytotoxicity for in vitro use. The first objective was to overcome the frequent restriction that high transfection efficiency is limited by the cytotoxicity of the non-viral carrier. We explored the potential of utilizing LPEIs with a molecular weight (MW) ranging from 1.0 to 9.5 kDa to overcome this limitation (Chapter 2). This study envisioned the decoupling of transfection efficiency from cytotoxicity and demonstrated that LPEIs with low MW are more efficient and significantly less toxic than their high MW counterparts in CHO-K1 cells in vitro. A follow-up study focused on the uptake and intracellular stability of various LPEI - polyplexes and plasmid DNA in order to investigate differences relevant for their efficacy. Furthermore, the influence of the ionic strength of the polyplex formation medium and presence of serum during the transfection process on the transfection efficiency was evaluated (Chapter 3). The results showed that we have a very robust system for LPEI-based nucleic acid delivery that is widely independent of external influences and suitable for routine transfection experiments. The interaction of polymer and plasmid DNA in living cells was additionally evaluated by fluorescence resonance energy transfer (FRET) -based techniques (Chapter 4). Furthermore, we hypothesized that a bioreversible crosslinking of low MW LPEIs would raise the polymer�s efficacy due to the higher MW, while the biodegradable linkages would undergo intracellular breakdown and hence minimize toxicity (Chapter 5). Our results proved the hypothesis: the most effective biodegradable PEI (LR-PEI) was capable of gene delivery in 27-fold more cells than the non-degradable starting material and moreover, the maximum transfection efficiency achieved in CHO-K1 cells had a value of about 73%, which is much higher than that of the LPEI derivative of similar MW. These biodegradable PEIs can be counted among the most effective polymer-based gene delivery vehicles, because the efficacy was nearly 3-fold higher than with BPEI 25 kDa and LPEI 22 kDa, to which newly synthesized polymers are usually compared. LR-PEI-mediated gene delivery was dependent on intracellular reduction and could be modulated by manipulation of the number of stabilizing bonds. After biodegradable PEI-based polyplexes had successfully been applied for the transfection of various cell lines in vitro, the next step was to evaluate their gene transfer ability in human primary cells and in a �hard-to-transfect� cell line (Chapter 6). In dendritic cells the transfection efficiency was nearly negligible and also accompanied by a remarkable toxicity. Human chondrocytes were more susceptible to PEI-based transfection, but the efficacy was much lower compared to the cell lines tested in Chapter 5. Last mentioned, we showed that the PEI backbone is a necessary prerequisite for efficient polymer-based gene delivery by the investigation of per-N-methylated PEI (Chapter 7). References: 1. Marshall E. Science 2002; 298: 510-511. 2. Sadelain M. Gene Ther 2004; 11: 569-573. 3. Boussif O, Lezoualc'h F, Zanta MA, Mergny MD, Scherman D, Demeneix B, Behr J. Proc Natl Acad Sci U S A 1995; 92: 7297-7301

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

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

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

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 mu m. 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 (similar to 2.4-fold), SC cells (similar to 1.4-fold) and fibroblasts (similar to 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

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

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 (SiNP). 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

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

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