Enzyme stability in nanoparticle preparations part 1: Bovine serum albumin improves enzyme function

Enzymes have gained attention for their role in numerous disease states, calling for research for their efficient delivery. Loading enzymes into polymeric nanoparticles to improve biodistribution, stability, and targeting in vivo has led the field with promising results, but these enzymes still suffer from a degradation effect during the formulation process that leads to lower kinetics and specific activity leading to a loss of therapeutic potential. Stabilizers, such as bovine serum albumin (BSA), can be beneficial, but the knowledge and understanding of their interaction with enzymes are not fully elucidated. To this end, the interaction of BSA with a model enzyme B-Glu, part of the hydrolase class and linked to Gaucher disease, was analyzed. To quantify the natural interaction of beta-glucosidase (B-Glu,) and BSA in solution, isothermal titration calorimetry (ITC) analysis was performed. Afterwards, polymeric nanoparticles encapsulating these complexes were fully characterized, and the encapsulation efficiency, activity of the encapsulated enzyme, and release kinetics of the enzyme were compared. ITC results showed that a natural binding of 1:1 was seen between B-Glu and BSA. Complex concentrations did not affect nanoparticle characteristics which maintained a size between 250 and 350 nm, but increased loading capacity (from 6% to 30%), enzyme activity, and extended-release kinetics (from less than one day to six days) were observed for particles containing higher B-Glu:BSA ratios. These results highlight the importance of understanding enzyme:stabilizer interactions in various nanoparticle systems to improve not only enzyme activity but also biodistribution and release kinetics for improved therapeutic effects. These results will be critical to fully characterize and compare the effect of stabilizers, such as BSA with other, more relevant therapeutic enzymes for central nervous system (CNS) disease treatments

Optimization of an Injectable Hydrogel Depot System for the Controlled Release of Retinal-Targeted Hybrid Nanoparticles

A drawback in the development of treatments that can reach the retina is the presence of barriers in the eye that restrain compounds from reaching the target. Intravitreal injections hold promise for retinal delivery, but the natural defenses in the vitreous can rapidly degrade or eliminate therapeutic molecules. Injectable hydrogel implants, which act as a reservoir, can allow for long-term drug delivery with a single injection into the eye, but still suffer due to the fast clearance of the released drugs when traversing the vitreous and random diffusion that leads to lower pharmaceutic efficacy. A combination with HA-covered nanoparticles, which can be released from the gel and more readily pass through the vitreous to increase the delivery of therapeutic agents to the retina, represents an advanced and elegant way to overcome some of the limitations in eye drug delivery. In this article, we developed hybrid PLGA-Dotap NPs that, due to their hyaluronic acid coating, can improve in vivo distribution throughout the vitreous and delivery to retinal cells. Moreover, a hydrogel implant was developed to act as a depot for the hybrid NPs to better control and slow their release. These results are a first step to improve the treatment of retinal diseases by protecting and transporting the therapeutic treatment across the vitreous and to improve treatment options by creating a depot system for long-term treatments

Microfluidic technology for the production of hybrid nanomedicines

Microfluidic technologies have recently been applied as innovative methods for the production of a variety of nanomedicines (NMeds), demonstrating their potential on a global scale. The capacity to precisely control variables, such as the flow rate ratio, temperature, total flow rate, etc., allows for greater tunability of the NMed systems that are more standardized and automated than the ones obtained by well-known benchtop protocols. However, it is a crucial aspect to be able to obtain NMeds with the same characteristics of the previously optimized ones. In this study, we focused on the transfer of a production protocol for hybrid NMeds (H-NMeds) consisting of PLGA, Cholesterol, and Pluronic® F68 from a benchtop nanoprecipitation method to a microfluidic device. For this aim, we modified parameters such as the flow rate ratio, the concentration of core materials in the organic phase, and the ratio between PLGA and Cholesterol in the feeding organic phase. Outputs analysed were the chemico–physical properties, such as size, PDI, and surface charge, the composition in terms of %Cholesterol and residual %Pluronic® F68, their stability to lyophilization, and the morphology via atomic force and electron microscopy. On the basis of the results, even if microfluidic technology is one of the unique procedures to obtain industrial production of NMeds, we demonstrated that the translation from a benchtop method to a microfluidic one is not a simple transfer of already established parameters, with several variables to be taken into account and to be optimized

Tween® preserves enzyme activity and stability in PLGA nanoparticles

Enzymes, as natural and potentially long-term treatment options, have become one of the most sought-after pharmaceutical molecules to be delivered with nanoparticles (NPs); however, their instability during formulation often leads to underwhelming results. Various molecules, including the Tween® polysorbate series, have demonstrated enzyme activity protection but are often used uncontrolled without optimization. Here, poly(lactic-co-glycolic) acid (PLGA) NPs loaded with β-glucosidase (β-Glu) solutions containing Tween® 20, 60, or 80 were compared. Mixing the enzyme with Tween® pre-formulation had no effect on particle size or physical characteristics, but increased the amount of enzyme loaded. More importantly, NPs made with Tween® 20:enzyme solutions maintained significantly higher enzyme activity. Therefore, Tween® 20:enzyme solutions ranging from 60:1 to 2419:1 mol:mol were further analyzed. Isothermal titration calorimetry analysis demonstrated low affinity and unquantifiable binding between Tween® 20 and β-Glu. Incorporating these solutions in NPs showed no effect on size, zeta potential, or morphology. The amount of enzyme and Tween® 20 in the NPs was constant for all samples, but a trend towards higher activity with higher molar rapports of Tween® 20:β-Glu was observed. Finally, a burst release from NPs in the first hour with Tween®:β-Glu solutions was the same as free enzyme, but the enzyme remained active longer in solution. These results highlight the importance of stabilizers during NP formulation and how optimizing their use to stabilize an enzyme can help researchers design more efficient and effective enzyme loaded NPs

Cholesterol-loaded nanoparticles ameliorate synaptic and cognitive function in Huntington's disease mice

Brain cholesterol biosynthesis and cholesterol levels are reduced in mouse models of Huntington's disease (HD), suggesting that locally synthesized, newly formed cholesterol is less available to neurons. This may be detrimental for neuronal function, especially given that locally synthesized cholesterol is implicated in synapse integrity and remodeling. Here, we used biodegradable and biocompatible polymeric nanoparticles (NPs) modified with glycopeptides (g7) and loaded with cholesterol (g7-NPs-Chol), which per se is not blood-brain barrier (BBB) permeable, to obtain high-rate cholesterol delivery into the brain after intraperitoneal injection in HD mice. We report that g7-NPs, in contrast to unmodified NPs, efficiently crossed the BBB and localized in glial and neuronal cells in different brain regions. We also found that repeated systemic delivery of g7-NPs-Chol rescued synaptic and cognitive dysfunction and partially improved global activity in HD mice. These results demonstrate that cholesterol supplementation to the HD brain reverses functional alterations associated with HD and highlight the potential of this new drug-administration route to the diseased brain

Preparation and optimization of PIT solid lipid nanoparticles via statistical factorial design.

The objective of this study was the preparation, physico-chemical characterization and statistical optimization of cationic solid lipid nanoparticles (SLN) prepared by the PIT method as potential carrier for gene therapy, emphasizing the application of factorial design in such a kind of studies. The preliminary screening from a physico-chemical point of view on three cationic lipids (CTAB, DDAB and DOTAP), selected on the basis of their different chemical structure and increasing lipophilicity, allowed us to select SLN with DOTAP, due to its higher zeta potential and smaller particle size. Afterward, a 2(2) full factorial experimental design was developed in order to study the effects of two independent variables (amount of DOTAP and concentration of lipid matrix) and their interaction on mean particle size and zeta potential values. The factorial planning was validated by ANOVA analysis; the correspondence between the predicted values of size and zeta and those measured experimentally confirmed the validity of the design and the equation applied for its resolution. The factorial design showed a significant influence of the independent variables on the selected parameters; in particular, a higher effect of DOTAP was observed on zeta potential value. Different dilutions of the optimized SLN containing 7% w/w of cutina CP and 1% w/w of DOTAP, with size and zeta potential values respectively of 462.9 nm and 50.8 mV, were in vitro examined to evaluate the possible cytotoxicity on two models of cell cultures: human prostate cancer androgen-non-responsive DU-145 cells and primary cultures of rat astrocytes