Antonio Di Martino

Amphiphilic chitosan-grafted-functionalized polylactic acid based nanoparticles as a delivery system for doxorubicin and temozolomide co-therapy

The aim of this work was to investigate the potential of an amphiphilic system comprising chitosan-grafted polylactide and carboxyl-functionalized polylactide acid as a carrier for the controlled release and co-release of two DNA alkylating drugs: doxorubicin and temozolomide. Polylactide and carboxyl-functionalized polylactide acid were obtained through direct melt polycondensation reaction, using methanesulfonic acid as a non-toxic initiator, and subsequently these were grafted to the chitosan backbone through a coupling reaction, utilizing 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide as a condensing agent. ATR-FTIR analysis and conductometric titration confirmed that a reaction between CS and PLA, PLACA2% and PLACA5% occurred. Chitosan-grafted-polylactide and polylactide-citric acid nanoparticles were prepared via the polyelectrolyte complex technique, applying dextran sulphate as a polyanion, and loaded with doxorubicin and temozolomide. The diameter of particles, ζ-potential and their relationship to temperature and pH were analysed in all formulations. Encapsulation, co-encapsulation efficiency and release studies were conducted in different physiological simulated environments and human serum. Results showed the continuous release of drugs without an initial burst in different physiological media.

Chitosan grafted low molecular weight polylactic acid for protein encapsulation and burst effect reduction

Chitosan and chitosan-grafted polylactic acid as a matrix for BSA encapsulation in a nanoparticle structure were prepared through a polyelectrolyte complexation method with dextran sulfate. Polylactic acid was synthetized via a polycondensation reaction using the non-metal-based initiator methanesulfonic acid and grafted to the chitosan backbone by a coupling reaction, with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as the condensing agent. The effect of concentration of the polymer matrix utilized herein on particle diameter, ζ-potential, encapsulation efficiency, and the release kinetic of the model protein bovine serum albumin at differing pH levels was investigated. The influence of pH and ionic strength on the behavior of the nanoparticles prepared was also researched. Results showed that grafting polylactic acid to chitosan chains reduced the initial burst effect in the kinetics of BSA release from the structure of the nanoparticles. Furthermore, a rise in encapsulation efficiency of the bovine serum albumin and diminishment in nanoparticle diameter were observed due to chitosan modification. The results suggest that both polymers actually show appreciable encapsulation efficiency; and release rate of BSA. CS-g-PLA is more suitable than unmodified CS as a carrier for controlled protein delivery.

Recognition of concanavalin A by cationic glucosylated liposomes.

The specificity of carbohydrate-lectin interaction has been reported as an attractive strategy for drug delivery in cancer therapy because of the high levels of lectins in several human malignancies. A novel cationic glucosylated amphiphile was therefore synthesized, as a model system, to attribute specificity toward d-glucose receptors to liposome formulations. Fluorescence experiments demonstrated that the monomeric glucosylated amphiphile is capable of interacting with fluorescently labeled concanavalin A, a D-glucose specific plant lectin. The interaction of concanavalin A with liposomes composed of a phospholipid and the glucosylated amphiphile was demonstrated by agglutination observed by optical density and dynamic laser light scattering measurements, thus paving the way to the preparation of other glycosilated amphiphiles differing for the length of polyoxyethylenic spacer, the sugar moieties, and/or the length of the hydrophobic chain.

Polysaccharide-based nanocomplexes for co-encapsulation and controlled release of 5-Fluorouracil and Temozolomide.

Polysaccharide-based nanocomplexes, intended for simultaneous encapsulation and controlled release of 5-Fluorouracil (5-FU) and Temozolomide (TMZ) were developed via the complexation method using chitosan, alginic and polygalacturonic acid. Investigation focused on the influence of polysaccharides on the properties of the system and amelioration of the stability of the drugs, in particular TMZ. The dimensions of particles and their ζ-potential were found to range between 100 and 200nm and -25 to +40mV, respectively. Encapsulation efficiency varied from 16% to over 70%, depending on the given system. The influence of pH on the release and co-release of TMZ and 5-FU was evaluated under different pH conditions. The stability of the loaded drug, in particular TMZ, after release was evaluated and confirmed by LC-MS analysis. Results suggested that the amount of loaded drug(s) and the release rate is connected with the weight ratio of polysaccharides and the pH of the media. One-way ANOVA analysis on the obtained data revealed no interference between the drugs during the encapsulation and release process, and in particular no hydrolysis of TMZ occurred suggesting that CS-ALG and CS-PGA would represent interesting carriers for multi-drug controlled release and drugs protection.

Organic-inorganic hybrid nanoparticles controlled delivery system for anticancer drugs

The use of organic-inorganic hybrid nanocarriers for controlled release of anticancer drugs has been gained a great interest, in particular, to improve the selectivity and efficacy of the drugs. In this study, iron oxide nanoparticles were prepared then surface modified via diazonium chemistry and coated with chitosan, and its derivative chitosan-grafted polylactic acid. The purpose was to increase the stability of the nanoparticles in physiological solution, heighten drug-loading capacity, prolong the release, reduce the initial burst effect and improve in vitro cytotoxicity of the model drug doxorubicin. The materials were characterized by DLS, ζ-potential, SEM, TGA, magnetization curves and release kinetics studies. Results confirmed the spherical shape, the presence of the coat and the advantages of using chitosan, particularly its amphiphilic derivative, as a coating agent, thereby surpassing the qualities of simple iron oxide nanoparticles. The coated nanoparticles exhibited great stability and high encapsulation efficiency for doxorubicin, at over 500μg per mg of carrier. Moreover, the intensity of the initial burst was clearly diminished after coating, hence represents an advantage of using the hybrid system over simple iron oxide nanoparticles. Cytotoxicity studies demonstrate the increase in cytotoxicity of doxorubicin when loaded in nanoparticles, indirectly proving the role played by the carrier and its surface properties in cell uptake.

Chitosan-based nanocomplexes for simultaneous loading, burst reduction and controlled release of doxorubicin and 5-fluorouracil

In this work, nanocomplexes based on chitosan grafted by carboxy-modified polylactic acid (SPLA) were prepared with the aim of loading simultaneously two anticancer drugs - doxorubicin and 5-fluorouracil, as well as to control their release, reduce the initial burst and boost cytotoxicity. The SPLA was prepared by a polycondensation reaction, using pentetic acid as the core molecule, and linked to the chitosan backbone through a coupling reaction. Nanocomplexes loaded with both drugs were formulated by the polyelectrolyte complexation method. The structure of the SPLA was characterized by 1H NMR, while the product CS-SPLA was analyzed by FTIR-ATR to prove the occurrence of the reaction. Results showed that the diameters and ζ-potential of the nanocomplexes fall in the range 120-200nm and 20-37mV, respectively. SEM and TEM analysis confirmed the spherical shape and dimensions of the nanocomplexes. The presence of hydrophobic side chain SPLA did not influence the encapsulation efficiency of the drugs but strongly reduced the initial burst and prolonged release over time compared to unmodified chitosan. MS analysis showed that no degradation or interactions between the drugs and carrier were exhibited after loading or 24h of release had taken place, confirming the protective role of the nanocomplexes. In vitro tests demonstrated an increase in the cytotoxicity of the drugs when loaded in the prepared carriers.

Role of the hydrophilic spacer of glucosylated amphiphiles included in liposome formulations in the recognition of Concanavalin A.

The functionalization of liposomes with glycosylated amphiphiles is an optimal strategy for targeted drug delivery, leading to enhanced efficacy as well as to reduced side effects of drugs. In fact, the presence of natural or synthetic glycolipids in vesicle formulations might increase their specificity toward lectins, a class of non-enzymatic sugar-binding proteins involved in cellular recognition and adhesion. The capability of a new glucosylated synthetic amphiphile to interact with Concanavalin A (Con A), a plant lectin used as model system, was investigated by a synergic experimental and computational approach, both as pure component and in formulation with a natural phospholipid. The comparison of the affinity with Con A of the new glucosylated amphiphile with respect to that of a previously described structural analogue demonstrates that the hydrophilic spacer length controls the exposure of the glucose residue on liposome surface, and consequently the recognition by the lectin.

A DSC investigation on the influence of gemini surfactant stereochemistry on the organization of lipoplexes and on their interaction with model membranes.

Previous investigations showed that the extent of DNA condensation and the efficiency in the transfection of liposomes formulated with 1,2-dimyristoyl-sn-glycero-phosphocholine and cationic stereomeric gemini surfactants depend heavily on the stereochemistry of the gemini. The influence of the stereochemistry on the interaction of lipoplexes with zwitterionic and anionic cell membrane models was investigated by differential scanning calorimetry to rationalize their different biological behavior. Further, the thermotropic behavior of the corresponding liposomes and of the spontaneous self-assemblies of gemini surfactants in the presence and in the absence of DNA was evaluated to correlate the physicochemical properties of lipoplexes and the stereochemistry of the cationic component. The obtained results show that the stereochemistry of the gemini surfactant controls lipoplexes organization and their mode and kinetic of interaction with different cell membrane models.

Enhancement of 5-aminolevulinic acid phototoxicity by encapsulation in polysaccharides based nanocomplexes for photodynamic therapy application

Polysaccharides based nanocomplexes have been developed for encapsulation, controlled delivery and to enhance the phototoxicity of the photosensitizer 5-aminolevulinic acid for application in photodynamic therapy. The nanocomplexes were prepared by coacervation in a solvent free environment using chitosan as polycation while alginic and polygalacturonic acid as polyanions. The complexes showed average dimension in the range 90-120nm, good stability in simulated physiological media and high drug encapsulation efficiency, up to 800μg per mg of carrier. Release studies demonstrate the possibility to tune the overall release rate and the intensity of the initial burst by changing the external pH. Cytotoxicity and photocytotoxicity tests confirmed the not toxicity of the used polysaccharides. Cell viability results confirmed the improvement of 5-aminolevulinic acid phototoxicity when loaded into the carrier compared to the free form. No effect of the irradiation on the nanocomplexes structure and on the release kinetics of the drug was observed. The results demonstrate that the prepared formulations have suitable properties for future application in photodynamic therapy and to ameliorate the therapeutic efficacy and overcome the side-effects related to the use of the photosensitizer 5-aminolevulinic acid.

Branched poly (lactic acid) microparticles for enhancing the 5-aminolevulinic acid phototoxicity

An innovative microcarrier based on a carboxy-enriched and branched polylactic acid derivative was developed to enhance the in vitro phototoxicity of the photosensitizer and prodrug 5-aminolevulinic. Microparticles, prepared by double emulsion technique and loaded with the prodrug were carefully characterized and the effect of the polymer structure on the chemical, physical and biological properties of the final product was evaluated. Results showed that microparticles have a spherical shape and ability to allocate up to 30 μg of the photosensitizer per mg of carrier despite their difference in solubility. Release studies performed in various simulated physiological conditions demonstrate the influence of the branched structure and the presence of the additional carboxylic groups on the release rate and the possibility to modulate it. In vitro assays conducted on human epithelial adenocarcinoma cells proved the not cytotoxicity of the carriers in a wide range of concentrations. The hemocompatibility and surface proteins adsorption were evaluated at different microparticles concentrations to evaluate the safety and estimate the possible microparticles residential time in the bloodstream. The advantages, of loading 5-aminolevulinic acid in the prepared carrier has been deeply described in terms of enhanced phototoxicity, compared to the free 5-aminolevulinic acid formulation after irradiation with light at 635 nm. The obtained results demonstrate the advantages of the prepared derivative compared to the linear polylactide for future application in photodynamic therapy based on the photosensitizer 5-aminolevulinic acid.