Retinal Delivery of the Protein Kinase C-ß Inhibitor Ruboxistaurin Using Non-Invasive Nanoparticles of Polyamidoamine Dendrimers.

Ruboxistaurin (RBX) is an anti-vascular endothelial growth factor (anti-VEGF) agent that is used in the treatment of diabetic retinopathy and is mainly given intravitreally. To provide a safe and effective method for RBX administration, this study was designed to develop RBX nanoparticles using polyamidoamine (PAMAM) dendrimer generation 5 for the treatment of diabetic retinopathy. Drug loading efficiency, and in vitro release of proposed complexes of RBX: PAMAM dendrimers were determined and the complexation ratio that showed the highest possible loading efficiency was selected. The drug loading efficiency (%) of 1:1, 2.5:1, and 5:1 complexes was 89.2%, 96.4%, and 97.6%, respectively. Loading capacities of 1:1, 2.5:1, and 5:1 complexes were 1.6%, 4.0%, and 7.2% respectively. In comparison, the 5:1 complex showed the best results in the aforementioned measurements. The in vitro release studies showed that in 8 h, the RBX release from 1:1, 2.5:1, and 5:1 complexes was 37.5%, 35.9%, and 77.0%, respectively. In particular, 5:1 complex showed the highest drug release. In addition, particle size measurements showed that the diameter of empty PAMAM dendrimers was 214.9 ± 8.5 nm, whereas the diameters of loaded PAMAM dendrimers in 1:1, 2.5:1, 5:1 complexes were found to be 461.0 ± 6.4, 482.4 ± 12.5, and 420.0 ± 7.1 nm, respectively. Polydispersity index (PDI) showed that there were no significant changes in the PDI between the free and loaded PAMAM dendrimers. The zeta potential measurements showed that the free and loaded nanoparticles possessed neutral charges due to the presence of anionic and cationic terminal structures. Furthermore, the safety of this formulation was apparent on the viability of the MIO-M1 cell lines. This nanoformulation will improve the therapeutic outcomes of anti-VEGF therapy and the bioavailability of RBX to prevent vision loss in patients with diabetic retinopathy.

Phosphate Dysregulation and Neurocognitive Sequelae.

The endocrine regulator proteins, fibroblast growth factor 23 (FGF23) and Klotho have been well studied as mediators of phosphate metabolism. FGF23 has been implicated in the renal excretion of phosphate by limiting the docking of sodium-dependent phosphate transporters, Npt2a and Npt2c, into the luminal side of renal proximal tubular epithelial cells. By limiting Npt2a/c activity in the renal tubular epithelial cells, phosphate is reabsorbed at lower rates and is excreted at higher rates. The action of Klotho is relatively less understood but has been implicated as an FGF23 cofactor in receptor binding. Klotho is mostly synthesized in the distal tubules of the nephron relative to FGF23's activity in proximal renal tubules. The neurological sequelae due to alterations in the FGF23-Klotho axis may be explained by the direct effects of these phosphate-regulating proteins on neuronal tissues or by the roles of these proteins in phosphate metabolism. Hyperphosphatemia has been associated with vascular wall stiffness that may alter blood flow and weakenvessels in the brain. In contrast, hypophosphatemia may alter ATP usage and metabolism in the central nervous system (CNS), leading to neurological compromise. Altered levels of FGF23 and Klotho have both been associated with neurocognitive decline, clinical dementia, memory loss, and poor executive function in humans. Furthermore, FGF23 and Klotho dysregulation has been linked to structural and functional changes of the cardiovascular system with an increased risk of stroke. Subsequent research should focus on characterizing the neuropathology associated with alterations in the FGF23-Klotho system and dysregulated phosphate metabolism.

Colored Polymeric Nanofiber Loaded with Minoxidil Sulphate as Beauty Coverage and Restoring Hair Loss.

Polymeric nanofibers fabricated by electrospinning either blank (PVA) or loaded with minoxidil sulphate have yielded optimum fibers with an average diameter 273 nm, and 511 nm, respectively. Thermal analysis of nanofibers indicated no chemical interaction. The NMR spectrum confirmed stability of nanofiber as there were no interactions between functional groups. Prepared nanofibers showed a 47.4% encapsulation efficiency and 73% yield. In vitro drug release of minoxidil sulphate from nanofiber exhibited an initial burst release followed by a slower release pattern. Stability studies revealed that minoxidil nanofiber was stable if stored at room temperature and protected from light with only loss of 9.6% of its nominal concentration within 6 months. As a result, the prepared solid/colored formula serves as an ideal formulation for such instable drug in liquid formula taking the advantage of the attractiveness of beauty colored coverage, and the simple, and non-tousled application.

Spatiotemporal PFQNM visualization of the effect of suicide dendriplexes on dividing HeLa cells.

Suicide gene delivery is significant in cancer therapy but has not been fully investigated on a cellular scale. Here, Peak Force Quantitative Nanomechanical atomic force microscopy (PFQNM-AFM) was applied to visualize the effect of herpes simplex virus thymidine kinase dendriplexes (G4AcFaHSTK) on the morphological and nanomechanical properties of individual live and dividing HeLa cells. Cells were then exposed to G4AcFaHSTK, followed by ganciclovir, and directly imaged by real-time PFQNM-AFM. Cell membrane liquefaction, cytoplasmic shrinkage, and cytoskeleton structure loss were observed during cell division. The average Young's modulus of the nuclear region increased with time as the cell continued from metaphase (6.29 kPa) to telophase (13.6 kPa) and then decreased (2.25 kPa) upon apoptosis. In contrast, cells exposed to either ganciclovir or G4AcFaHSTK alone have no changes. Thus, understanding the real-time effects of suicide dendriplexes on the cytoskeletal and nanomechanical behaviors of cancer cells may provide new methods for cancer treatment.

Visualizing the attack of RNase enzymes on dendriplexes and naked RNA using atomic force microscopy.

Cationic polymers such as poly(amidoamine), PAMAM, dendrimers have been used to electrostatically complex siRNA molecules forming dendriplexes for enhancing the cytoplasmic delivery of the encapsulated cargo. However, excess PAMAM dendrimers is typically used to protect the loaded siRNA against enzymatic attack, which results in systemic toxicity that hinders the in vivo use of these particles. In this paper, we evaluate the ability of G4 (flexible) and G5 (rigid) dendrimers to complex model siRNA molecules at low +/- ratio of 2/1 upon incubation for 20 minutes and 24 hours. We examine the ability of the formed G4 and G5 dendriplexes to shield the loaded siRNA molecules and protect them from degradation by RNase V1 enzymes using atomic force microscopy (AFM). Results show that G4 and G5 dendrimers form similar hexagonal complexes upon incubation with siRNA molecules for 20 minutes with average full width of 43±19.3 nm and 62±8.3 at half the maximum height, respectively. AFM images show that these G4 and G5 dendriplexes were attacked by RNase V1 enzyme leading to degradation of the exposed RNA molecules that increased with the increase in incubation time. In comparison, incubating G4 and G5 dendrimers with siRNA for 24 hours led to the formation of large particles with average full width of 263±60 nm and 48.3±2.5 nm at half the maximum height, respectively. Both G4 and G5 dendriplexes had a dense central core that proved to shield the loaded RNA molecules from enzymatic attack for up to 60 minutes. These results show the feasibility of formulating G4 and G5 dendriplexes at a low N/P (+/-) ratio that can resist degradation by RNase enzymes, which reduces the risk of inducing non-specific toxicity when used in vivo.

AFM analysis of C60 and a poly(amido amine) dendrimer-C60 nanoconjugate.

Atomic force microscopy (AFM) was used to study the nanoscopic structure and topography of buckminsterfullerene (C60) and a conjugate of C60 with generation four, amine-terminated, poly(amido amine) dendrimer (PAMAM-G4). The conjugate contains a PAMAM-G4 core and C60 shell formed by reacting PAMAM-G4 with an excess of C60. Fractal patterns of C60 were observed in nanoscopic AFM images when solutions of different concentrations of C60 in pyridine or toluene were dried at room temperature. In contrast, no fractal patterns were detected in the AFM images of the dendrimer-C60 nanoconjugate, prepared from pyridine solution in a similar manner. Thus, the C60-shell alone is not sufficient to impart the same fractal patterns on the conjugate.

Direct real-time molecular scale visualisation of the degradation of condensed DNA complexes exposed to DNase I.

The need to protect DNA from in vivo degradation is one of the basic tenets of therapeutic gene delivery and a standard test for any proposed delivery vector. The currently employed in vitro tests, however, presently provide no direct link between the molecular structure of the vector complexes and their success in this role, thus hindering the rational design of successful gene delivery agents. Here we apply atomic force microscopy (AFM) in liquid to visualise at the molecular scale and in real time, the effect of DNase I on generation 4 polyamidoamine dendrimers (G4) complexed with DNA. These complexes are revealed to be dynamic in nature showing a degree of mobility, in some cases revealing the addition and loss of dendrimers to individual complexes. The formation of the G4-DNA complexes is observed to provide a degree of protection to the DNA. This protection is related to the structural morphology of the formed complex, which is itself shown to be dependent on the dendrimer loading and the time allowed for complex formation.