RICERCA ITALIANA     

Research program

Multiscale modelling and development of process reactors for polymeric nanoparticle precipitation

University Co-ordinator

Università degli Studi di TORINO - SCIENZA E TECNOLOGIA DEL FARMACO - ()

Research Unit Leader

Franco Dosio

Description

The advantages of using nanoparticulate carriers in the clinical setting may be many and significant: the method enables substances with very low water solubility to be administered; it improves biodistribution in the host and provides controlled release of the pharmacological agent, increasing the therapeutic index through an improvement in the amount of drug able to reach the specific target. Clearly this brings with it a reduction in side effects.
Furthermore, the efficacy of colloidal systems in IV administration may be limited if these are recognised by macophages of the reticuloendothelial system (RES). Thus both the type of polymer used for the matrix (lattice) or capsule, in which the active agent is dissolved, and the polymer/external interface behaviour, are of considerable importance.
To increase the blood survival time, the nanoparticle surface must be shielded with highly hydrophilic, flexible and non-ionic polymers, such as polyethylene glycols (PEG). Such shielded nanoparticles have been shown to selectively extravasate into disease sites, such as tumours, where the vascular endothelium is more permeable and lymphatic drainage is reduced.
In this research programme we will use polymers based on poly cyanoacrylate, and in particular amphiphilic, stable but biodegradable and biocompatible copolymers. The main chain (skeleton) is made of cyanoacrylate with branched arms having different characteristics (hydrophobicity, steric hindrance).
The starting point will be a copolymer used in our laboratory: poly(methoxy polyethylene glycol cyanoacrylate-co-hexadecyl cyanoacrylate) (poly(MePEGCA-co-HDCA), in which the PEG chains alternate with hexadecyl chains. During nanoparticle production (known as nanoprecipitation) the lateral chains of this amphiphilic polymer move in different directions depending on their polarity: the hydrophobic hexadecyl chains form the particle's solid core whereas the PEG chains move outwards towards the aqueous phase.
Furthermore, it is possible to activate the top of the flexible PEG arm with molecules that have specific receptors in biological systems.

A) Synthesis of polymers useful for nanoparticles preparation.
The first step will be the synthesis of cyanoacetic esters of hexadecanol, monomethoxy polyethylene glycol (MePEG) and ter-butyloxycarbonyl-amino-PEG (t-Boc-HNPEG) by esterification of cyanoacetic acid with the corresponding alcohols. The reaction will be performed in dichloromethane with dicyclohexylcarbodiimides and dimethylaminopyridine as catalysts. Polymers poly-hexadecyl-cyanoacetate (PHDCA), and copolymers with PEG derivatives poly(MePEGCA-co-HDCA) and poly(H2NPEGCA-co-HDCA), will be obtained by condensation/polymerisation of the relative monomers in ethanol as solvent, in the presence of formaldehyde and dimethylamine or pyrrolidine as catalyst.
The project will analyse the importance of the alkyl chain length (with more than 16 carbon atoms to increase lipophilicity, or with fewer than 16 carbons to reduce it). In this way it will be possible to change the ratio between hydrophilic/hydrophobic components of the copolymer and thus its physico/chemical properties after incorporation of the active principle. Furthermore, the biological behaviour of the nanoparticle will be changed. The synthesized polymers will then be fully characterised in terms of structure, molecular weight, ratio of monomers and purity, by NMR and GPC (gel permeation chromatography) techniques.
The optimized and validate synthetic method will be shared with the other research units in order to prepare large amount of polymers (tens-to-hundreds of grams). In this way these polymers will be used by different research units, employing different methodologies for nanoparticle preparation.

B) Preparation of nanoparticles.
The nanoparticles will be prepared with a nanoprecipitation method: the pre-shaped polymer dissolved in an appropriate solvent (mixable with water) such as acetone, ethanol, tetrahydrofurane etc.. is mixed with a large amount of water in the presence of a surfactant, under vigorous stirring.
This method is based on dissolution of the polymer (insoluble in water) in an organic solvent, followed by addition of an aqueous solution containing a surfactant agent. Different operational parameters are important, including the polymer concentration ratio between solvents and water. In our previous research the best concentration was found to be 10 mg/ml, with a 1:2 ratio (acetone:water). In these conditions the nanospheres formed immediately by polymer precipitation, due to diffusion of the organic solvent into the aqueous phase. Addition of the organic solution in which the polymer is dissolved creates a diffusion front of the solvent, along which there is a marked reduction in interfacial tension. As a consequence, polymer solubility is reduced and it precipitates along the diffusion front, naturally generating spherical matrices. The next step involves concentration and filtration of the suspension on microfilters.
To prepare nanocapsules, a small amount of oil is added to the organic phase in order to obtain an oil/water emulsion. The polymer precipitates on the emulsion interface.
Another significant advantage in the use of amphiphilic polymers such as poly(MePEGCA-co-HDCA) or its analogues is that the presence of surfactants is not necessary. Surfactants are frequently toxic, allergenic and difficult to eliminate from suspensions.
The project will improve and extend studies regarding:
The phenomenon of interfacial polymerisation
Selection of the best operative conditions to dissolve polymers
Production of nanoparticles in quantity, employing different procedures (described by other research units)
Evaluation of the stability of the batches obtained and analysis of the byproducts
All batches of the prepared nanoparticles will be characterised in full from the physico/chemical standpoint: dimensional analysis, formulation stability, morphologic evaluation (SEM, TEM), surface charge and hydrophobicity (by zeta potential analysis and hydrophobic interaction chromatography).
The characteristics of the nanoparticles obtained with poly(MePEGCA-co-HDCA) may be compared with those obtained with other polymers without the PEG moiety (such as poly(hexadecylcyanoacrylate) (PHDCA) and derivatives with branches of different lengths.


C) Preparation of nanoparticles containing pharmacologically-active molecules.
The drug of choice will be doxorubicin. Doxorubicin hydrochloride belongs to the general group of chemotherapy drugs known as anthracycline antibiotics. It is used to treat a large variety of tumors: non–Hodgkin’s lymphoma, multiple myeloma, acute leukemias, and cancers of the breast, endometrium, lung, and ovary. The molecule is lipophilic, flexible and has intrinsic fluorescence. This latter characteristic can be used in biological tests to monitor the progress of drug or nanoparticles containing it, by microscope fluorescence.
Encapsulation of doxorubicin in poly(MePEGCA-co-HDCA) nanospheres has been achieved through two different protocols: dissolving the drug in the aqueous phase followed by nanoprecipitation; dissolving the drug in triethylamine in the organic phase followed by nanoprecipitation
All nanoparticle preparation phases involving the presence of antitumour drugs will be done respecting safety standards for workers.