Unraveling Polymeric Nanoparticles Cell Uptake Pathways: Two Decades Working to Understand Nanoparticles Journey to Improve Gene Therapy.

Polymeric nanoparticles have aroused an increasing interest in the last decades as novel advanced delivery systems to improve the treatment of many diseases. Hard work has been performed worldwide designing and developing polymeric nanoparticles using different building blocks, which target specific cell types, trying to avoid bioaccumulation and degradation pathways. The main handicap of the design is to understand the final fate and the journey that the nanoparticle will follow, which is intimately ligated with the chemical and physical properties of the nanoparticles themselves and specific factors of the targeted cells. Although the huge number of published scientific articles regarding polymeric nanoparticles for biomedical applications, their use in clinics is still limited. This fact could be explained by the limited data reporting the interaction of the huge diversity of polymeric nanoparticles with cells. This knowledge is essential to understand nanoparticle uptake and trafficking inside cells to the subcellular target structure.In this chapter, we aim to contribute to this field of knowledge by: (1) summarizing the polymeric nanoparticles properties and cellular factors that influence nanoparticle endocytosis and (2) reviewing the endocytic pathways classified as a function of nanoparticle size and as a function of the receptor playing a role. The revision of previously reported endocytic pathways for particular polymeric nanoparticles could facilitate scientist involved in this field to easily delineate efficient delivery systems based on polymeric nanoparticles.

Development of an optimized freeze-drying protocol for OM-PBAE nucleic acid polyplexes.

Long-term stability of polyplexes used for biomedical purposes is an objective envisaged by any research group developing this kind of nanoformulations. However, since biodegradable polymers such as oligopeptide end-modified poly (ß-aminoester) (OM-PBAE) are frequently used to ensure safety, and formulations are produced as aqueous dispersions, the stability of the nanoformulations is usually compromised. In this context, freeze-drying has aroused as a promising storage alternative to obtain solid nanoformulations with enhanced stability over time. Lyophilization is a challenging step that usually produces aggregation. Although some studies already achieved freeze-dried PBAE nanoparticles, none of them detailed the parameters that are critical for the success of this process. Moreover, due to the specific composition of each formulation, the critical parameters for the correct freeze-drying process need to be adjusted for each polyplex developed. In this paper, we have studied the variables that have a direct influence on the manufacturing and lyophilization of OM-PBAE nanoparticles with the aim to develop a versatile and robust freeze-drying receipt that properly preserves the library of polyplexes designed in our group, which have different pKa depending on the modification applied.

Design of parenteral MNP-loaded PLGA nanoparticles by a low-energy emulsification approach as theragnostic platforms for intravenous or intratumoral administration.

Encapsulation of magnetic nanoparticles (MNP) into PLGA nanoparticles has been achieved by nano-emulsion templating using for the first time both, a low-energy emulsification method as well as biocompatible components accepted for pharmaceuticals intended for human use. The incorporation of MNP by nano-emulsion templating method proposed in this work has been investigated in two different systems applying mild process conditions and is shown to be simple and versatile, providing stable MNP-loaded PLGA nanoparticles with tunable size and MNP concentration. MNP-loaded PLGA nanoparticles showed sizes below 200nm by DLS and 50nm by TEM, and mean MNP loading per PLGA nanoparticle of 1 to 4, depending on the nanoparticle dispersion composition. Physical-chemical features suggest that the MNP-loaded PLGA nanoparticles obtained are good candidates for intravenous or intratumoral administration.

PLGA nanoparticles from nano-emulsion templating as imaging agents: Versatile technology to obtain nanoparticles loaded with fluorescent dyes.

The interest in polymeric nanoparticles as imaging systems for biomedical applications has increased notably in the last decades. In this work, PLGA nanoparticles, prepared from nano-emulsion templating, have been used to prepare novel fluorescent imaging agents. Two model fluorescent dyes were chosen and dissolved in the oil phase of the nano-emulsions together with PLGA. Nano-emulsions were prepared by the phase inversion composition (PIC) low-energy method. Fluorescent dye-loaded nanoparticles were obtained by solvent evaporation of nano-emulsion templates. PLGA nanoparticles loaded with the fluorescent dyes showed hydrodynamic radii lower than 40nm; markedly lower than those reported in previous studies. The small nanoparticle size was attributed to the nano-emulsification strategy used. PLGA nanoparticles showed negative surface charge and enough stability to be used for biomedical imaging purposes. Encapsulation efficiencies were higher than 99%, which was also attributed to the nano-emulsification approach as well as to the low solubility of the dyes in the aqueous component. Release kinetics of both fluorescent dyes from the nanoparticle dispersions was pH-independent and sustained. These results indicate that the dyes could remain encapsulated enough time to reach any organ and that the decrease of the pH produced during cell internalization by the endocytic route would not affect their release. Therefore, it can be assumed that these nanoparticles are appropriate as systemic imaging agents. In addition, in vitro toxicity tests showed that nanoparticles are non-cytotoxic. Consequently, it can be concluded that the preparation of PLGA nanoparticles from nano-emulsion templating represents a very versatile technology that enables obtaining biocompatible, biodegradable and safe imaging agents suitable for biomedical purposes.

Galantamine-loaded PLGA nanoparticles, from nano-emulsion templating, as novel advanced drug delivery systems to treat neurodegenerative diseases.

Polymeric nanoparticles could be promising drug delivery systems to treat neurodegenerative diseases. Among the various methods of nanoparticle preparation, nano-emulsion templating was used in the present study to prepare galantamine-loaded nano-emulsions by a low-energy emulsification method followed by solvent evaporation to obtain galantamine-loaded polymeric nanoparticles. This approach was found to be suitable because biocompatible, biodegradable and safe nanoparticles with appropriate features (hydrodynamic radii around 20 nm, negative surface charge and stability higher than 3 months) for their intravenous administration were obtained. Encapsulation efficiencies higher than 90 wt% were obtained with a sustained drug release profile as compared to that from aqueous and micellar solutions. The enzymatic activity of the drug was maintained at 80% after its encapsulation into nanoparticles that were non-cytotoxic at the required therapeutic concentration. Therefore, novel galantamine-loaded polymeric nanoparticles have been designed for the first time using the nano-emulsification approach and showed the appropriate features to become advanced drug delivery systems to treat neurodegenerative diseases.

PLGA nanoparticles prepared by nano-emulsion templating using low-energy methods as efficient nanocarriers for drug delivery across the blood-brain barrier.

Neurodegenerative diseases have an increased prevalence and incidence nowadays, mainly due to aging of the population. In addition, current treatments lack efficacy, mostly due to the presence of the blood-brain barrier (BBB) that limits the penetration of the drugs to the central nervous system. Therefore, novel drug delivery systems are required. Polymeric nanoparticles have been reported to be appropriate for this purpose. Specifically, the use of poly-(lactic-co-glycolic acid) (PLGA) seems to be advantageous due to its biocompatibility and biodegradability that ensure safe therapies. In this work, a novel approximation to develop loperamide-loaded nanoparticles is presented: their preparation by nano-emulsion templating using a low-energy method (the phase inversion composition, PIC, method). This nano-emulsification approach is a simple and very versatile technology, which allows a precise size control and it can be performed at mild process conditions. Drug-loaded PLGA nanoparticles were obtained using safe components by solvent evaporation of template nano-emulsions. Characterization of PLGA nanoparticles was performed, together with the study of the BBB crossing. The in vivo results of measuring the analgesic effect using the hot-plate test evidenced that the designed PLGA loperamide-loaded nanoparticles are able to efficiently cross the BBB, with high crossing efficiencies when their surface is functionalized with an active targeting moiety (a monoclonal antibody against the transferrin receptor). These results, together with the nanoparticle characterization performed here are expected to provide sufficient evidences to end up to clinical trials in the near future.