Luisa Tondelli

Poly(ethylene glycol) imidazolyl formates as oligomeric drug-binding matrices

Abstract Poly(ethylene glycol)s of molecular weight 200 and 1000 have been functionalized by reaction with N,N-carbonyldiimidazole. The resulting imidazolyl formates could be isolated in a pure state and proved to be able to react with model amines and hydroxylated compounds giving the corresponding urethanes or carbonates. These results indicate that the new activated matrices can be used for the preparation of oligomeric derivatives of aminated or hydroxylated drugs.

Induction of humoral and enhanced cellular immune responses by novel core-shell nanosphere- and microsphere-based vaccine formulations following systemic and mucosal administration

Anionic surfactant-free polymeric core-shell nanospheres and microspheres were previously described with an inner core constituted by poly(methylmethacrylate) (PMMA) and a highly hydrophilic outer shell composed of a hydrosoluble co-polymer (Eudragit L100-55). The outer shell is tightly linked to the core and bears carboxylic groups capable of adsorbing high amounts (antigen loading ability of up to 20%, w/w) of native basic proteins, mainly by electrostatic interactions, while preserving their activity. In the present study we have evaluated in mice the safety and immunogenicity of new vaccine formulations composed of these nano- and microspheres and the HIV-1 Tat protein. Vaccines were administered by different routes, including intramuscular, subcutaneous or intranasal and the results were compared to immunization with Tat alone or with Tat delivered with the alum adjuvant. The data demonstrate that the nano- and microspheres/Tat formulations are safe and induce robust and long-lasting cellular and humoral responses in mice after systemic and/or mucosal immunization. These delivery systems may have great potential for novel Tat protein-based vaccines against HIV-1 and hold promise for other protein-based vaccines.

Functional Polymeric Nano/Microparticles for Surface Adsorption and Delivery of Protein and DNA Vaccines

The use of particulate polymeric carriers holds great promise for the development of effective and affordable DNA and protein subunit vaccines. Rational development of such vaccine formulations requires a detailed understanding of their physico-chemical properties, cell-free and in vitro behaviour, in addition to particle uptake and processing mechanisms to antigen presenting cells capable of stimulating safe and effective immune responses. We here provide an overview on functional polymeric nano- and micro-particles designed for surface adsorption of proteins and DNA antigens currently under investigation for the formulation of new vaccines, including comments on their preparation method, antigen delivery strategy, cell-free and in vitro behaviour. In addition, we focus on their influence in activating antigen-specific humoral and/or cellular immune responses and on their potential for the development of new vaccines.

Core–shell microspheres by dispersion polymerization as drug delivery systems

Poly(methyl methacrylate) core-shell particles in the submicron scale range were prepared by dispersion polymerization through an appropriate selection of the experimental parameters and in particular of the initiator and stabilizer amount and the medium solvency power. Low initiator concentration, high steric stabilizer amount and a low solvency power medium must be employed. In these condi- tions, monosized particles of about 300 nm can be obtained in which the outer layer is constituted by the steric stabilizer, a commercial poly(methacrylate)-marked Eudragit E 100, which affords amino groups able to interact with biologically active compounds via specific or non-specific interactions.

Immunization with low doses of HIV-1 tat DNA delivered by novel cationic block copolymers induces CTL responses against Tat.

Cytotoxic T cell responses are key to the control of intracellular pathogens including HIV-1. In particular, HIV-1 vaccines based on regulatory proteins, such as Tat, are aimed at controlling HIV-1 replication and at blocking disease development by inducing cytotoxic T cell responses. Naked DNA is capable of inducing such responses but it requires several inoculations of high amounts of DNA, and/or prime-boost regimens. Here, we show that a novel class of cationic block copolymers protect the DNA from DNAse I digestion, and improve DNA delivery to antigen-presenting cells (APCs) after intramuscular (i.m.) vaccination. In particular, three cationic block copolymers (K1, K2 and K5) were used to deliver the HIV-1 pCV-tat DNA vaccine in BALB/c mice. The results indicate that vaccination with a very low dose (1 microg) of pCV-tat delivered by the cationic block copolymer K2 is safe and, as compared to naked DNA (up to 30 microg), greatly increases the CTL response against Tat, which was detected in all animals in the absence or in the presence of re-stimulation.

Micellar-type complexes of tailor-made synthetic block copolymers containing the HIV-1 tat DNA for vaccine application

A novel class of cationic block copolymers constituted by a neutral hydrophilic poly(ethylene glycol) (PEG) block and a positively charged poly(dimethylamino)ethyl methacrylate block was prepared for delivery of DNA. These block copolymers spontaneously assemble with DNA to give in aqueous medium micellar-like structures. Five of these novel block copolymers (K1-5), differing in the length of both the PEG chain and the linear charge density of the poly(dimethylamino)ethyl methacrylate block, were prepared and analyzed for gene delivery, gene expression and safety. All five block copolymers protected DNA from DNAse I digestion and delivered the DNA into the cell. However, only three of them (K1, K2 and K5) released the DNA at level allowing efficient gene expression into cells. No toxic effects of both the copolymers alone or their DNA complexes were observed in vitro or in mice. In addition, copolymers were scarcely immunogenic. These results indicate that this novel class of cationic block copolymers is safe and possesses the biological characteristics required for DNA delivery, thus, representing promising vehicles for DNA vaccination.

Core–shell microspheres by dispersion polymerization as promising delivery systems for proteins

Functional poly(methyl methacrylate) core–shell microspheres were prepared by dispersion polymerization. An appropriate selection of experimental parameters and in particular of the initiator and stabilizer amount and of the medium solvency power allowed a monodisperse sample as large as 600 nm to be prepared. To this purpose, low initiator concentration, high steric stabilizer amount and a low solvency power medium were employed. The microspheres present a core–shell structure in which the outer shell is constituted by the steric stabilizer which affords carboxylic groups able to interact with basic proteins, such as trypsin, whose adsorption is essentially driven by the carboxylic group density in the microsphere shell. Finally, fluorescent microspheres were prepared for biodistribution studies and shown to be readily taken up by the cells both in vitro and in vivo. These results suggest that these microspheres are promising delivery systems for the development of novel protein-based vaccines.

Complex associates of plasmid DNA and a novel class of block copolymers with PEG and cationic segments as new vectors for gene delivery.

Cationic block copolymers, consisting of a poly(ethylene glycol) block and a block deriving from the poly(dimethylamino)ethyl methacrylate were prepared via a two-step procedure, based on the use of macroinitiators. By appropriately changing the experimental conditions and reacting the poly(dimethylamino)ethyl methacrylate block with iodo- or bromo-alkyl derivatives, a variety of ionic block copolymers with tuned physicochemical properties were prepared. These block copolymers are able to spontaneously self-assemble with plasmid DNA to produce oriented and shielded vectors, with physicochemical properties appropriate for in vivo applications. In addition, the formation of a complex between the cationic block copolymer and the plasmid DNA results in a nuclease resistance increase due to the stable nature of the complex.

Priming with a very low dose of DNA complexed with cationic block copolymers followed by protein boost elicits broad and long-lasting antigen-specific humoral and cellular responses in mice.

Cationic block copolymers spontaneously assemble via electrostatic interactions with DNA molecules in aqueous solution giving rise to micellar structures that protect the DNA from enzymatic degradation both in vitro and in vivo. In addition, we have previously shown that they are safe, not immunogenic and greatly increased antigen-specific CTL responses following six intramuscular inoculations of a very low dose (1microg) of the vaccine DNA as compared to naked DNA. Nevertheless, they failed to elicit detectable humoral responses against the antigen. To gain further insight in the potential application of this technology, here we show that a shorter immunization protocol based on two DNA intramuscular inoculations of 1microg of DNA delivered by these copolymers and a protein boost elicits in mice broad (both humoral and cellular) and long-lasting responses and increases the antigen-specific Th1-type T cell responses and CTLs as compared to priming with naked DNA. These results indicate that cationic block copolymers represent a promising adjuvant and delivery technology for DNA vaccination strategies aimed at combating intracellular pathogens.

Tailor-made core-shell nanospheres for antisense oligonucleotide delivery: IV.Adsorption/release behaviour

The adsorption/release behaviour of oligodeoxynucleotides (ODNs) on double functional core-shell polymethylmethacrylate nanospheres, with a narrow size distribution, is described. The outer shell consists of alkyl or glycolic chains containing permanently-charged quaternary ammonium groups. Ion pair formation between negatively-charged ODN phosphate groups and positively-charged groups, present on the nanosphere surface, is the main mechanism of interaction. The amount of adsorbed ODN depends on both the ODN concentration and the nanosphere surface charge density. An adsorption-induced swelling mechanism is proposed in which a modification of the charged diffuse layer around the nanospheres increases the ODN binding site accessibility with increasing ODN concentration. Adsorption on the nanosphere surface prevents serum degradation of the ODNs. ODN release is negligible in the presence of culture medium but occurs gradually in the presence of serum. No significant cytotoxicity of the free nanoparticles was found in PBMC and CEM cells after 24 h at ODN concentrations required for antisense activity.