Chitosan nanoparticles, produced by ionic gelation, are among the most intensely studied nanosystems for drug delivery. However, a lack of inter-laboratory reproducibility and a poor physicochemical understanding of the process of particle formation have been slowing their potential market applications. To address these shortcomings, the current study presents a systematic analysis of the main polymer factors affecting the nanoparticle formation driven by an initial screening using systematic statistical Design of Experiments (DoE). In summary, we found that for a given chitosan to TPP molar ratio, the average hydrodynamic diameter of the particles formed is strongly dependent on the initial chitosan concentration. The degree of acetylation of the chitosan was found to be the second most important factor involved in the system’s ability to form particles. Interestingly, viscosimetry studies indicated that the particle formation and the average hydrodynamic diameter of the particles formed were highly dependent on the presence or absence of salts in the medium. In conclusion, we found that by controlling two simple factors of the polymer solution, namely its initial concentration and its solvent environment, it is feasible to control in a reproducible manner the production and characteristics of chitosan particles ranging in size from nano- to micrometres.

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Chitosans, β-1,4-linked partially N-acetylated linear polyglucosamines, are very versatile and promising functional biopolymers. Understanding their structure-function relationships requires sensitive and accurate structural analyses to determine parameters like degree of polymerization (DP), fraction of acetylation (F), or pattern of acetylation (P). NMR, the gold standard for F analysis, requires large amounts of sample. Here, we describe an enzymatic/mass spectrometric fingerprinting method to analyze the F of chitosan polymers. The method combines the use of chitinosanase, a sequence-specific hydrolase that cleaves chitosan polymers into oligomeric fingerprints, ultrahigh-performance liquid chromatography-electrospray ionization-mass spectrometry (UHPLC-ESI-MS), and partial least-squares regression (PLSR). We also developed a technique to simulate enzymatic fingerprints in silico that were used to build the PLS models for F determination. Overall, we found our method to be as accurate as NMR while at the same time requiring only microgram amounts of sample. Thus, the method represents a powerful technique for chitosan analysis.

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Recently we reported for the first time a new type of nanocapsules consisting of an oily core and a polymer shell made of a polyglutamic acid-polyethylene glycol (PEG-PGA) grafted copolymer with a 24% w/w PEG content. The goal of the work presented here has been to develop a new version of these nanocapsules, in which the shell is made of a di-block PEG-PGA copolymer with a 57% w/w PEG content and to evaluate their potential for improving the biodistribution and pharmacokinetics of the anticancer drug docetaxel (DCX). A comparative analysis of the biodistribution of fluorescently labeled PGA-PEG nanocapsules versus PGA nanocapsules or a control nanoemulsion (containing the same oil than the nanocapsules) showed that the nanocapsules, and in particular PEGylated nanocapsules, have significantly higher half-life, MRT (Mean Residence Time) and AUC (Area under the Curve) than the nanoemulsion. On a separate set of experiments, PGA-PEG nanocapsules were loaded with DCX and their antitumor efficacy was evaluated in a xenograft U87MG glioma mouse model. The results showed that the survival rate for mice treated with DCX-loaded nanocapsules was significantly increased over the control Taxotere®, while the antitumoral effect of both formulations was comparable (60% tumor growth inhibition with respect to the untreated mice). These results highlight the potential use of these novel nanocapsules as a new drug delivery platform in cancer therapy.

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Here, we report the in vivo proof of-concept of a novel nanocarrier, poly-l-asparagine (PASN) nanocapsules, as an anticancer targeted drug delivery system. The nanocapsules were loaded with the fluorescent marker DiD (1,1'-dioctadecyl-3,3,3',3'-tetramethylindodicarbocyanine perchlorate) and also with the model drug docetaxel to evaluate the biodistribution and efficacy profiles in healthy and glioma-bearing mice, respectively. Regardless of their cargo, the nanocapsules presented a size close to 180 nm, a surface charge around -40 mV and an encapsulation efficiency of 75-90%. The biodistribution study in healthy mice showed that PASN nanocapsules led to a two- and three-fold increment in the mean residence time (MRT) and area under the curve (AUC) values, respectively, compared to those of a non-polymeric nanoemulsion. Finally, the efficacy/toxicity study indicated that the encapsulated drug was as efficacious as the commercial formulation (Taxotere(®)), with the additional advantage of being considerably less toxic. Overall, these results suggest the potential of PASN nanocapsules as drug nanocarriers in anticancer therapy.