Robert Hennig

Ligand-functionalized nanoparticles target endothelial cells in retinal capillaries after systemic application

To date, diseases affecting vascular structures in the posterior eye are mostly treated by laser photocoagulation and multiple intraocular injections, procedures that destroy healthy tissue and can cause vision-threatening complications. To overcome these drawbacks, we investigate the feasibility of receptor-mediated nanoparticle targeting to capillary endothelial cells in the retina after i.v. application. Cell-binding studies using microvascular endothelial cells showed receptor-specific binding and cellular uptake of cyclo(RGDfC)-modified quantum dots via the αvβ3 integrin receptor. Conversely, Mueller cells and astrocytes, representing off-target cells located in the retina, revealed only negligible interaction with nanoparticles. In vivo experiments, using nude mice as the model organism, demonstrated a strong binding of the ligand-modified quantum dots in the choriocapillaris and intraretinal capillaries upon i.v. injection and 1-h circulation time. Nontargeted nanoparticles, in contrast, did not accumulate to a significant amount in the target tissue. The presented strategy of targeting integrin receptors in the retina could be of utmost value for future intervention in pathologies of the posterior eye, which are to date only accessible with difficulty.

Nanoparticle multivalency counterbalances the ligand affinity loss upon PEGylation

The conjugation of receptor ligands to shielded nanoparticles is a widely used strategy to precisely control nanoparticle-cell interactions. However, it is often overlooked that a ligands affinity can be severely impaired by its attachment to the polyethylene glycol (PEG) chains that are frequently used to protect colloids from serum protein adsorption. Using the model ligand EXP3174, a small-molecule antagonist for the angiotensin II receptor type 1 (AT1R), we investigated the ligands affinity before and after its PEGylation and when attached to PEGylated nanoparticles. The PEGylated ligand displayed a 580-fold decreased receptor affinity compared to the native ligand. Due to their multivalency, the nanoparticles regained a low nanomolar receptor affinity, which is in the range of the affinity of the native ligand. Moreover, a four orders of magnitude higher concentration of free ligand was required to displace PEGylated nanoparticles carrying EXP3174 from the receptor. On average, one nanoparticle was decorated with 11.2 ligand molecules, which led to a multivalent enhancement factor of 22.5 compared to the monovalent PEGylated ligand. The targeted nanoparticles specifically bound the AT1R and showed no interaction to receptor negative cells. Our study shows that the attachment of a small-molecule ligand to a PEG chain can severely affect its receptor affinity. Concomitantly, when the ligand is tethered to nanoparticles, the immense avidity greatly increases the ligand-receptor interaction. Based on our results, we highly recommend the affinity testing of receptor ligands before and after PEGylation to identify potent molecules for active nanoparticle targeting.

Nanoparticles for the treatment of ocular neovascularizations

Neovascular diseases of the posterior eye like age-related macular degeneration, proliferative diabetic retinopathy or retinopathy of prematurity carry a tremendous burden for patient and health care system alike. Although intravitreal injections of anti-VEGF based therapeutics have significantly improved the visual outcome for many patients, current therapeutic options still show significant drawbacks such as the injection-related risk of contracting an infection. Due to their ability to encapsulate drugs with otherwise poor bioavailability, accumulate in areas of increased vascular permeability and control the release of active ingredients over time, nanoparticle systems have been widely researched to enhance current therapeutic strategies and expand the therapeutic arsenal. In this review, emphasis is placed both on the possibilities and drawbacks that a systemic nanoparticle-based therapy could have in the context of neovascular posterior eye diseases. Recent investigations into intravenous and intravitreal administration of nanomaterials and their potential to deliver potent drugs and genes to pathologic lesions will also be presented. Furthermore, we will focus on the exceptional anti-oxidative and anti-angiogenic properties of selected nanoscale systems that carve out new paths for the treatment of these severe posterior eye diseases.

Kidney Podocytes as Specific Targets for cyclo(RGDfC)-Modified Nanoparticles

Renal nanoparticle passage opens the door for targeting new cells like podocytes, which constitute the exterior part of the renal filter. When cyclo(RGDfC)-modified Qdots are tested on isolated primary podocytes for selective binding to the αvβ3 integrin receptor a highly cell- and receptor-specific binding can be observed. In displacement experiments with free cyclo(RGDfC) IC(50) values of 150 nM for αvβ3 integrin over-expressing U87-MG cells and 60 nM for podocytes are measured. Confocal microscopy shows a cellular Qdot uptake into vesicle-like structures. Our ex vivo study gives clear evidence that, after renal filtration, nanoparticles can be targeted to podocyte integrin receptors in the future. This could be a highly promising approach for future therapy and diagnostics of podocyte-associated diseases.

Formation of fenestrae in murine liver sinusoids depends on plasmalemma vesicle-associated protein and is required for lipoprotein passage.

Liver sinusoidal endothelial cells (LSEC) are characterized by the presence of fenestrations that are not bridged by a diaphragm. The molecular mechanisms that control the formation of the fenestrations are largely unclear. Here we report that mice, which are deficient in plasmalemma vesicle-associated protein (PLVAP), develop a distinct phenotype that is caused by the lack of sinusoidal fenestrations. Fenestrations with a diaphragm were not observed in mouse LSEC at three weeks of age, but were present during embryonic life starting from embryonic day 12.5. PLVAP was expressed in LSEC of wild-type mice, but not in that of Plvap-deficient littermates. Plvap-/- LSEC showed a pronounced and highly significant reduction in the number of fenestrations, a finding, which was seen both by transmission and scanning electron microscopy. The lack of fenestrations was associated with an impaired passage of macromolecules such as FITC-dextran and quantum dot nanoparticles from the sinusoidal lumen into Disses space. Plvap-deficient mice suffered from a pronounced hyperlipoproteinemia as evidenced by milky plasma and the presence of lipid granules that occluded kidney and liver capillaries. By NMR spectroscopy of plasma, the nature of hyperlipoproteinemia was identified as massive accumulation of chylomicron remnants. Plasma levels of low density lipoproteins (LDL) were also significantly increased as were those of cholesterol and triglycerides. In contrast, plasma levels of high density lipoproteins (HDL), albumin and total protein were reduced. At around three weeks of life, Plvap-deficient livers developed extensive multivesicular steatosis, steatohepatitis, and fibrosis. PLVAP is critically required for the formation of fenestrations in LSEC. Lack of fenestrations caused by PLVAP deficiency substantially impairs the passage of chylomicron remnants between liver sinusoids and hepatocytes, and finally leads to liver damage.

Branched Polymer–Drug Conjugates for Multivalent Blockade of Angiotensin II Receptors

The use of angiotensin receptor blockers (ARBs) for treatment of ocular diseases associated with neovascularizations, such as proliferative diabetic retinopathy, shows tremendous promise but is presently limited due to short intravitreal half-life. Conjugation of ARB molecules to branched polymers could vastly augment their therapeutic efficacy. EXP3174, a potent non-peptide ARB, was conjugated to branched poly(ethylene glycol) (PEG) and poly(amido amine) (PAMAM) dendrimers: 7.8 ligand molecules were tethered to each 40 kDa PEG molecule whereas 16.7 ligand molecules were linked to each PAMAM generation 5 dendrimer. The multivalent PEG and PAMAM conjugates blocked AT1R signaling with an IC50 of 224 and 36.3 nM, respectively. The 6-fold higher affinity of the multivalent ligand-conjugated PAMAM dendrimers was due to their unique microarchitecture and ability to suppress polymer-drug interactions. Remarkably, both polymer-drug conjugates exhibited no cytotoxicity, in stark contrast to plain PAMAM dendrimers. With sufficiently long vitreous half-lives, both synthesized polymer-ARB conjugates have the potential to pave a new path for the therapy of ocular diseases accompanied by retinal neovascularizations.

Multivalent targeting of AT1 receptors with angiotensin II-functionalized nanoparticles

Abstract The angiotensin II receptor type 1 (AT1R) is a G protein-coupled receptor of paramount significance since it is overexpressed in a number of diseased tissues that are highly attractive for nanoparticle targeting. However, it is also expressed at physiological levels in healthy tissue. Multivalent interactions mediated by multiple AT1R-binding moieties per nanoparticle could promote a high binding avidity to AT1R overexpressing cells and concomitantly spare off-target tissue. To investigate the feasibility of this approach, angiotensin II was thiolated and conjugated to PEGylated quantum dots. Nanoparticle binding, uptake and affinity to several cell lines was investigated in detail. The colloids were rapidly taken up by clathrin-mediated endocytosis into AT1R-expressing cells and showed no interaction with receptor negative cells. The EC50 of the thiolated angiotensin II was determined to be 261 nM, whereas the ligand-conjugated Qdots activated the receptor with an EC50 of 8.9 nM. This 30-fold higher affinity of the nanoparticles compared to the unconjugated peptide clearly demonstrated the presence of multivalent effects when using agonist-targeted nanoparticles. Our study provides compelling evidence that, despite being immediately endocytosed, Ang II-coupled nanoparticles exert potent multivalent ligand–receptor interactions that can be used to establish high affinities to an AT1R overexpressing cell and tissue.

Cyclic RGD peptides target human trabecular meshwork cells while ameliorating connective tissue growth factor-induced fibrosis

Abstract The major risk factor for primary open-angle glaucoma is increased intraocular pressure stemming from elevated outflow resistance in the trabecular meshwork (TM) region. Integrins play a pivotal role in the TM by influencing its biological properties and growth factor signaling. Pathologic changes in the TM are partially mediated by growth factors like connective tissue growth factor (CTGF). Specific targeting of TM cells could play a critical clinical role by increasing the therapeutic efficacy of nanoparticles, e.g. for nonviral gene delivery. Quantum dots with cyclo(RGDfC) covalently immobilized to their surface effectively targeted cultured TM cells and were rapidly and efficiently endocytosed by binding to αvβ3 and αvβ5 integrins. Compared to the integrin-overexpressing U87-MG cell line, the association of RGD-modified nanoparticles with the TM cells was significantly higher. Binding and uptake into TM cells was receptor-mediated and suppressible with free peptide. Soluble cyclic RGD peptides effectively attenuated CTGF-mediated effects and inhibited CTGF signaling. Due to their antagonism for αvβ3 and αvβ5 integrins, these cyclic RGD pentapeptides effectively ameliorated the CTGF-induced effects and strongly promoted specific nanoparticle association. Thus, cyclic RGD peptides are powerful multifunctional ligands for both addressing nanomaterials to the TM and interfering with pathologic CTGF signaling upon arrival.

Multivalent nanoparticles bind the retinal and choroidal vasculature.

The angiotensin II receptor type 1 (AT1R), which is expressed in blood vessels of the posterior eye, is of paramount significance in the pathogenesis of severe ocular diseases such as diabetic retinopathy and age-related macular degeneration. However, small molecule angiotensin receptor blockers (ARBs) have not proven to be a significant therapeutic success. We report here on a nanoparticle system consisting of ARB molecules presented in a multivalent fashion on the surface of quantum dots (Qdots). As a result of the multivalent receptor binding, nanoparticles targeted cells with high AT1R expression and inhibited their angiotensin receptor signaling with an IC50 of 3.8 nM while showing only minor association to cells with low AT1R expression. After intravenous injection into the tail vein of mice, multivalent nanoparticles accumulated in retinal and choroidal blood vessels of the posterior eye. At the same time, multivalent ligand display doubled the Qdot concentration in the blood vessels compared to non-targeted Qdots. Remarkably, ARB-targeted Qdots showed no pronounced accumulation in AT1R-expressing off-target tissues such as the kidney. Following systemic application, this multivalent targeting approach has the potential to amplify AT1R blockade in the eye and concomitantly deliver a therapeutic payload into ocular lesions.