Mariano Licciardi

In this paper three copolymers of polyhydroxyethylaspartamide (PHEA), bearing in the side chains polyethylene glycol (PEG) and/or hexadecylamine (C(16)) (PHEA-PEG, PHEA-PEG-C(16) and PHEA-C(16) respectively) have been studied as potential colloidal drug carriers for ocular drug delivery. The physical characterization of all three PHEA derivatives, using the Langmuir trough (LT) and micellar affinity capillary electrophoresis (MACE) techniques allowed to assume that whereas alone PHEA backbone is an inert polymer with respect to the interactions with lipid membranes and drug complexation, when PHEA chains are grafted with long alkyl chains like C(16) or in combination C(16) chains and hydrophilic chains like PEG, copolymers with lipid membrane interaction ability and drug complexation capability are obtained. In vitro permeability studies performed on primary cultured rabbit conjunctival and corneal epithelia cells, using PHEA-C(16) and PHEA-PEG-C(16) as micelle carriers for netilmic...

ABSTRACT New cationic α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) graft copolymers were synthesized by ATRP, using diethylamino ethyl methacrylate (DEAEMA) as monomer for polymerization, yielding polycations (PHEA-pDEAEMA) able to condense DNA. Then, consecutive ATRP conditions were set up on PHEA-pDEAEMA to obtain copolymers containing also hydrophilic chains (PHEA-IB-pDMAEMA-pPEGMA) able to improve biocompatibility of polyplexes and to provide them stealth properties. Agarose gel studies showed that the copolymers effectively condensed plasmid DNA to form polyplexes. Light scattering studies were used to analyze the size and the f-potential of these polyplexes, showing that copolymers were able to condense the pDNA leading to the formation of nanoscale systems. The copolymers PHEAIB-pDEAEMA showed high cytocompatibility that was improved with the presence of PEGMA units in the side chain. The transfection efficiency (luciferase) of polyplexes obtained with all copolymers was evaluated on B16F10 cell line obtaining a moderate transfection efficiency in comparison with bPEI that can be explained, supposing a low release of pDNA from polyplexes at endocellular level.

A recombinant hybrid composed of the two major allergens of the Parietaria pollen Par j 1 and Par j 2 has been generated by DNA recombinant technology (PjED). This hybrid was produced in E. coli at high levels of purity. Then, the engineered derivative has been combined with a synthetic polyaminoacidic derivative having a poly(hydroxyethyl)aspartamide (PHEA) backbone and bearing both butyryl groups (C4) and succinyl (S) moieties in the side chain (PHEA-C4-S). The allergen-copolymer nanoaggregate was characterized by means of DLS, zeta potential, electrophoretic mobility and atom force microscopy analysis displaying the formation of a stable complex. Its safety has been proved in vitro on a murine cell line, human erythrocytes and basophils. Moreover, the formation of the complex did not alter the ability of the allergens to cross-link surface bound specific IgE demonstrating that the combination of an engineered hybrid with a copolymer did not interfere with its biological activity ...

ABSTRACT In order to obtain new advanced functional materials capable of recognizing drug molecules, the polybenzofulvene backbone of molecular brush poly-6-MOEG-9-TM-BF3k has been functionalized with a “synthetic dynamic receptor” composed of two 1-adamantyl-urea moieties linked together by means of a dipropyleneamino bridge as in Meijer’s bis(adamantylurea) pincer (BAUP). This functional material bearing synthetic receptors potentially capable of recognizing/loading and then delivering drug molecules, was used to prepare colloidal drug delivery systems (by means of soft-interaction with BAUP) for delivering the model anti-cancer drug doxorubicin. The resulting nanostructured drug delivery systems containing the physically loaded drug were characterized in terms of drug loading and release, dimensions and zeta potential, and in vitro cell activity and uptake on two different cell lines (i. e. the human bronchial epithelial 16HBE and the human colon cancer HCT116). On normal cells, free DOXO resulted more cytotoxic than DOXO-loaded nanogels at the higher tested concentration and, only on cancer cells, DOXO-loaded nanogels show similar or slightly higher cytotoxicity values than free DOXO, suggesting potential advantages in the treatment of cancer. These results were supported by fluorescence microscopy studies, which suggested that DOXO-loaded nanogels provide an extracellular reservoir of the drug, which is gradually released and internalized within the cells.

In this study, a polybenzofulvene derivative named poly-6-MOEG-9-BF3k, was evaluated as polymeric material for the production of injectable thermoresponsive nano-aggregates able to load low molecular weight peptidic drug, like the anticancer leuprolide. Thermoresponsive behavior of poly-6-MOEG-9-BF3k was studied in aqueous media by evaluating scattering intensity variations by means of DLS in function of temperature. Zeta potential measurements and SEM observations were also carried out. Moreover, critical aggregation temperature of the poly-6-MOEG-9-BF3k polymer was evaluated by pyrene fluorescence analysis. Then, the ability of prepared thermoresponsive aggregates to protect this model oligopeptide drug and regulate its release rate in function of external temperature was evaluated in vitro. Finally, biocompatibility of poly-6-MOEG-9-BF3k aggregates was tested in vitro on a healthy cell line (human bronchial epithelial cell; 16-HBE) and in vivo on rat animal model upon subcutaneous administration.

The main objective of this study was to evaluate the ability of folic acid-functionalized diblock copolymer micelles to improve the delivery and uptake of two poorly water-soluble anti-tumor drugs, tamoxifen and paclitaxel, to cancer cells through folate receptor targeting. The diblock copolymer used in this study comprised a hydrophilic poly[2-(methacryloyloxy)ethyl phosphorylcholine] (MPC) block, carrying at the chain end the folate targeting moiety, and a pH-sensitive hydrophobic poly[2-(diisopropylamino)ethyl methacrylate] (DPA) block (FA-MPC-DPA). The drug-loading capacities of tamoxifen- and paclitaxel-loaded micelles were determined by high performance liquid chromatography and the micelle dimensions were determined by dynamic light scattering and transmission electron microscopy. Cell viability studies were carried out on human chronic myelogenous leukaemia (K-562) and colon carcinoma cell lines (Caco-2) in order to demonstrate that drug-loaded FA-MPC-DPA micelles exhibited ...

Hydrogels seem to be one of the most promising classes of modern biomaterials for the development of components of devices related to therapy and diagnosis. The main areas of today's application of hydrogels as biomaterials include: synthetic wound care coverings (hydrogels ...

To prepare new copolymers, useful for gene delivery, based on alpha, beta-poly-(N-2-hydroxyethyl)-D, L-aspartamide (PHEA) as a polymeric backbone and bearing an oligoamine such as diethylenetriamine in the side chain. Moreover, in order to reduce solvent volume and make the reaction faster, microwave-assisted heating was used. PHEA copolymers bearing different amounts of diethylenetriamine were prepared using bis(4-nitrophenyl) carbonate as a condensing agent with the use of microwaves. Chemical, physico-chemical and biological characterization of PHEA-diethylenetriamine copolymers and their complexes obtained with DNA were performed. Copolymers showed good DNA complexing and condensing abilities depending on the oligoamine derivatization degree and good hemocompatibility. Moreover, plasmid DNA/copolymer polyplexes showed very good cytocompatibility on B16F10 and N2A cell lines. Results support the use of these copolymers as gene delivery systems in the future. Finally, the use of microwaves makes the proposed synthetic method advantageous as time and solvents are saved.

The aim of this work was to produce copolymers with an appropriate hydrophilic/hydrophobic balance able to form nanoaggregates with protein molecules and to be used as ideal materials in the field of oral peptide/protein delivery. New anionic polymers obtained by the conjugation of carboxy-bearing ligands, like succinic anhydride and/or cysteine, to hydrophobized α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) copolymers have been synthesized and characterized. Starting copolymer was synthesized by the partial derivatization of hydroxyl groups on the PHEA backbone with butylamine (C4) (obtaining the PHEA-C4 copolymer, bearing a butyl moiety). The consecutive reaction of PHEA-C4 with succinic anhydride permitted the PHEA-C4-S copolymer to be obtained, bearing pendant carboxylic groups as well. Finally, part of the pendant carboxylic groups were conjugated to cysteine via an amidic bond, obtaining PHEA-C4-S-Cyst. All synthesized copolymers, PHEA-C4, PHEA-C4-S, and PHEA-C4-S-Cyst, exhibit the ability to interact with insulin in aqueous medium forming nanoaggregates. Physical characterization of prepared insulin/copolymer nanoaggregates was carried out by means of turbidimetric measurements and DLS analysis. These studies demonstrated that synthesized copolymers form colloidal aggregates in the presence of insulin, with size ranging between 62 and 216 nm. Stability studies in the presence of the peptidase α-chymotrypsin showed also the ability of synthesized copolymers to increase insulin stability against enzymatic degradation in the order PHEA-C4-S-Cyst > PHEA-C4-S > PHEA-C4. Moreover, in dosage form such as tablets, the synthesized copolymers displayed the properties to prolong disintegration time and control release of the embedded peptide drug into media mimicking intestinal fluids. The administration of insulin in the presence of PHEA-C4-S-Cyst and PHEA-C4-S copolymers resulted in the ability to provoke a certain absorption of insulin and consequently to induce in vivo hypoglycemic effects on rats after oral administration with respect to free insulin. In particular, the hypoglycemic effect shown by PHEA-C4-S/insulin nanoaggregates was equal to almost 30% of the effect observed after the administration of insulin by conventional subcutaneous administration and about 20% in the case of PHEA-C4-S-Cyst/insulin nanoaggregates. These copolymers are good starting materials for the preparation of an oral dosage form of proteins.

In this paper the preparation of magnetic nanocarriers (MNCs), containing superparamagnetic domains, is reported, useful as potential magnetically targeted drug delivery systems. The preparation of MNCs was performed by using the PHEA-IB-p(BMA) graft copolymer as coating material through the homogenization-solvent evaporation method. Magnetic and nonmagnetic nanocarriers containing flutamide (FLU-MNCs) were prepared. The prepared nanocarriers have been exhaustively characterized by dynamic light scattering (DLS), transmission electron microscopy (TEM), and magnetic measurements. Biological evaluation was performed by in vitro cytotoxicity and cell uptake tests and in vivo biodistribution studies. Magnetic nanocarriers showed dimensions of about 300 nm with a narrow size distribution, an amount of loaded FLU of 20% (w/w), and a superparamagnetic behavior. Cell culture experiments performed on prostate cancer cell line LNCaP demonstrated the cytotoxic effect of FLU-MNCs. In vivo biodistribution studies carried out by the application of an external magnetic field in rats demonstrated the effect of the external magnet on modifying the biodistribution of FLU-MNCs. FLU-MNCs resulted efficiently internalized by tumor cells and susceptible to magnetic targeting by application of an external magnetic field. The proposed nanocarriers can represent a very promising approach to obtain an efficient magnetically targeted anticancer drug delivery system.

A simple and efficient method for the synthesis of polyaspartamide-based brush copolymers using Atom Transfer Radical Polymerization (ATRP) is here presented. The syntheses were performed by using two subsequent steps. In the first step the macroinitiator was obtained ...

A new PHEA-IB-PMANa(+) copolymer has been synthesized and its pH-induced self-assembly has been investigated in an aqueous medium. PHEA-IB-PMANa+ formed nanoparticles with diameters from 25 to 50 nm upon protonation of the carboxylic acid moieties dislocated along the grafted polymethacrylate sodium salt side chains. The physico-chemical characterization of the nanoparticles was performed using light scattering, zeta-potential measurements, SEM, and AFM. Doxorubicin-loaded nanoparticles were prepared and drug release profiles were evaluated under conditions mimicking physiological media. A biological characterization was carried out by testing the cytotoxicity on Caco-2 cells, and cellular uptake on mouse monocyte macrophage (J774 A.1) and Caco-2 cells.

Aim of this paper was that to prepare biocompatible, polyaspartamide based copolymers containing spermine or spermine/hydrophobic side chains able to condense nucleic acids and to transfect mammalian cells. Copolymers were prepared starting from alpha,beta-poly-(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) and exploiting the reactive hydroxyl groups in the polymeric side chains by subsequent activation reactions to obtain PHEA-Spermine (PHEA-Spm) and PHEA-Spermine-Butyramide (PHEA-Spm-C(4)). Molecular, physico-chemical and biological characterization of copolymers and interpolyelectrolyte complexes with plasmid DNA was performed. Experimental results evidenced that these copolymers are able to form complexes with plasmid DNA already at low polycation/DNA weight ratio ranging from 0.75/1 to 2/1. Interpolyelectrolyte complexes with decreased size were obtained when increasing the polycation/DNA weight ratio, until nanosized dimensions were reached. Copolymers as well as complexes were not haemolytic and non toxic in vitro. In vitro cell transfection with PHEA derivatives showed good biocompatibility and high transfection efficiency (luciferase) in cancer cells in comparison with commercially available, but toxic transfection agents.

Novel polyaspartamide non-viral carriers for gene therapy were synthesized by introducing, on the same polymer backbone, positively charged groups, for electrostatic interactions with DNA, and thiol groups for the formation of disulfide bridges between polymer chains. The introduction of thiols was aimed to have a vector with low redox potential sensitivity: disulfide crosslinking in fact, being stable in extracellular environment, allowed either to have stable complexes in plasma, that can protect DNA from metabolism, or to be reduced inside the cell, where the excess of glutathion in reduced form maintains a low redox potential. The consequent destabilization of the complex after disulfide cleavage can release DNA selectively inside the cells. Alpha,beta-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was used as starting polymer being a highly water-soluble synthetic polymer, already proposed with success as therapeutic carrier by our group. In this study, PHEA was firstly functionalised with ethylendiamine, obtaining a well defined copolymer with pendant primary amine groups (PHEA-EDA), to which N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) were linked in two subsequent steps, allowing the introduction of thiol and cationic groups respectively. Finally DTT treatment lead to the final PHEA-EDA-SH-CPTA thiopolycation, named PESC. The present work describes the synthesis and characterization of the thiopolycation PESC. 1H NMR spectroscopy detected the derivatization molar degrees in SPDP and CPTA; the formation of DNA complexes (thiopolyplexes), their stability in the presence of polyanions and the ability to release DNA under reductive conditions were studied by agarose gel electrophoresis. DNase II degradation study was carried out to detect the ability of thiopolyplex to stabilize DNA towards enzymatic metabolism. Thiopolyplexes were then characterized by Dynamic Light Scattering (DLS) and Zeta Potential analysis. Finally, in vitro toxicity profile (MTT) and gene transfer efficiency (Luciferase assay) were carried out to evaluate thiopolyplex biocompatibility, safety and efficacy to be used as gene delivery system.

Poly(hydroxyethylaspartamide) (PHEA) derivatives bearing at the polyaminoacidic backbone poly(ethyleneglycol) (2000 or 5000 Da) or both poly(ethyleneglycol) and hexadecylalkylamine as pendant moieties were investigated as polymeric colloidal drug carriers. The ability of the PHEA derivatives to solubilize hydrophobic drugs was investigated using paclitaxel, amphotericin B and methotrexate. The results demonstrated that the drug solubility depends on both macromolecule composition and drug physicochemical properties. In particular, PEG/hexadecylalkylamine co-grafting increased significantly the solubilization properties of PHEA for the considered drugs while the conjugation of PEG only did not endow PHEA with drug carrier properties. A stability study carried out with paclitaxel/PHEA-PEG(5000)-hexadecylalkylamine demonstrated that the drug/carrier system is characterized by physicochemical instability, which is strictly related to the incubation pH. However, the carrier was found to partially prevent drug degradation. Investigations performed using murine myeloid leukaemia NFS-60 cell line showed that paclitaxel loaded PHEA-PEG(5000)-hexadecylalkylamine possesses high pharmacological activity with IC(50) value of 22.3 ng/ml. Pharmacokinetic studies carried out by intravenous administration of paclitaxel loaded PHEA-PEG(5000)-hexadecylalkylamine to Balb/c mice demonstrated that the carrier modifies the in vivo paclitaxel fate. In particular, PHEA-PEG(5000)-hexadecylalkylamine prolonged the drug distribution and elimination phase of 6 and 17 times, respectively; in addition, it increased the systemic availability (AUC) by about 30 times.

A family of graft copolymers derivatives obtained from α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) have been studied as potential self-assembling macromolecules forming stable polymeric micelles at low critical micellar concentration. These polymers are obtained grafting on PHEA poly(ethylene glycol) (PEG) (Mw 5000 g/mol) (PHEA–PEG), hexadecylamine (PHEA–C16) or both moieties (PHEA–PEG–C16). The PHEA derivatives were characterised by a multi-angle light scattering (MALS) photometer on line

In situ gel forming systems have been prepared by linking polylactic acid (PLA) to a water soluble and polyfunctional polymer, such as α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA). Three graft copolymers PHEA–PLA with a different derivatization degree in PLA, have been synthesized and characterized. PHEA–PLA graft copolymer with the highest amount in PLA has been used to prepare solutions in organic solvents able to

The non-viral gene vector properties of a protein-like polymer, the α, β-poly (N-2-hydroxyethyl)-d, l-aspartamide (PHEA) were investigated after its derivatization with 3-(carboxypropyl) trimethyl-ammonium chloride (CPTA) as molecule bearing cationic ...

Salmon calcitonin (sCT) is characterized by a poor oral availability. A new copolymer, β-poly(N-2-hydroxyethyl)-graft-{N-2-ethylene[2-poly(methacrylic acid sodium salt)isobutyrate]}-d,l-aspartamide (PHEA-IB-p(MANa(+))), was designed for the oral administration of sCT through the formation of supramolecular aggregates (SAs) based on electrostatic interactions. Several sCT/PHEA-IB-p(MANa(+)) weight ratios were characterized by turbidimetry, DLS, zeta potential, and microscopy analysis. After the incubation of sCT/PHEA-IB-p(MANa(+)) complex with digestive enzymes, 10% (w/w) of loaded sCT was released in the native form. In vitro investigation was carried out to determine the copolymer effect on the permeability of sCT in Caco-2 cell monolayers. sCT pharmacokinetic profile and the pharmacodynamic effect on calcium plasma level were determined following an oral administration of the lead sCT/PHEA-IB-p(MANa(+)) SA (1/5 ratio) in rats. The SA yielded a marked prolongation of the sCT lowering calcium effect. The maximum decrease, 35% with respect the basal calcium plasma level at time 0 h, was achieved after 4h post-administration, and after 7 h, a decrease of 20% was still present. Differently, sCT yielded a transient calcium decrease that was completely restored after 5h. The higher bioavailability of sCT administered as SA was confirmed by the pharmacokinetic studies. In fact, the AUC and the Cmax were about 15 times higher for the sCT formulated as SA than the free sCT. This study indicates the potentials of PHEA-IB-p(MANa(+)) as carrier of sCT for oral delivery.

Paclitaxel was attached to poly(hydroxyethylaspartamide) via a succinic spacer arm by a two-step protocol: (1) synthesis of 2'-O-succinyl-paclitaxel; (2) synthesis of PHEA-2'-O-succinyl-paclitaxel. The 2'-O-succinyl-paclitaxel derivative and the macromolecular conjugate were characterized by UV, IR, NMR and mass spectrometry analysis. The reaction yields were over 95% and the purity of products over 98%. Paclitaxel release and degradation from 2'-O-succinyl-paclitaxel occurred at a faster rate at pH 5.5 than 7.4. After 30 h of incubation at pH 5.5 and 7.4 the released free paclitaxel was about 40 and 20%, respectively. In plasma both drug release and degradation were found to occur at a higher rate than in buffer at pH 7.4 suggesting that an enzymatic mechanism could be involved. The paclitaxel release and degradation from PHEA-2'-O-succinyl-paclitaxel were negligible at pH 5.5 and 7.4 and very slow in plasma. Investigation carried out using murine myeloid cell line showed that the polymeric prodrug maintains partial pharmacological activity of paclitaxel. The DL50 of the conjugate (over 40 ng/ml) as compared to free paclitaxel (about 1 ng/ml) was correlated to the slow drug release. Finally a pharmacokinetic study carried out by intravenous inoculation of the macromolecular prodrug to mice demonstrated that the polymer conjugation modify dramatically the in vivo fate of the drug. The conjugate disappeared from the bloodstream much more quickly as compared to both free drug and naked polymer. Massive accumulation of bioconjugate in the liver (80% of the dose) was found to persist throughout 1 week.

The functionalization of α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) with glycidyl methacrylate (GMA) gives rise to a water-soluble copolymer PHEA-GMA (PHG) containing double bonds and ester groups in the side chain. Aqueous solutions of PHG alone or in combination with N,N′ methylenbisacrylamide (BIS) have been exposed to a γ-ray source at different irradiation doses in order to obtain polymeric networks. All samples have been

In this work, an efficient method for the synthesis of hyaluronic acid based brush copolymers using atom transfer radical polymerization (ATRP) has been reported. At first, two different hyaluronic acid (HA) based macroinitiators have been prepared and then they have been used for the polymerization via ATRP of hydrophilic or hydrophobic molecules carrying vinyl portions. In particular, by linking 2-bromo-2-methylpropionic acid (BMP) to the primary hydroxyl groups of tetrabutyl ammonium salt of HA (HA-TBA) or to amino groups of the ethylenediamino derivative of HA-TBA (HA-TBA-EDA), two macroinitiators (HA-TBA-BMP and HA-TBA-EDA-BMP) have been obtained. Then they have been used for the ATRP of poly(ethylene glycol) methacrylate (PEGMA), butyl methacrylate (BUTMA) or N-isopropylacrylamide (NIPAM) using a complex of Cu(I) and 2,2'-Bipyridyl (Bpy), as a catalyst. Both macroinitiators and final copolymers, named as HA-BMP-pPEGMA, HA-BMP-pBUTMA, HA-BMP-pNIPAM, HA-EDA-BMP-pPEGMA, HA-EDA-BMP-pBUTMA and HA-EDA-BMP-pNIPAM, have been characterized by spectroscopic analysis and size exclusion chromatography to confirm the success of the polymerization process.

The clinical usefulness of antitumor chemotherapy has been strongly limited by the lack of specificity of most anticancer drugs, which act also against healthy cells. The aim of this work was to design, synthesize, and evaluate a macromolecular prodrug of Cytarabine, a known antitumor drug, which is a specific substrate for plasmin enzyme whose concentration is high in various kinds of tumor mass as a result of plasminogen activator secretion. alpha,beta-Poly(N-hydroxyethyl)-DL-aspartamide (PHEA), a known synthetic and biocompatible polyamino acid, was used as a drug carrier, and Cytarabine was linked to PHEA by D-Val-Leu-Lys spacer synthesized beginning from Cbz-D-Val-LeuOH dipeptide and N6-CbzLys methyl ester. The content of Cytarabine in the purified PHEA-D-Val-Leu-Lys-Cytarabine conjugate was equal to 3% w/w. In vitro experiments in the presence of plasmin evidenced the ability of this enzyme to strongly increase drug release from the macromolecular prodrug, as well as plasma incubation shows high stability of drug-polymer linkage. The direct linkage of Cytarabine to PHEA was also performed and, like PHEA-D-Val-Leu-Lys-Cytarabine conjugate, the obtained PHEA-Cytarabine conjugate showed high stability in plasma, but no release of Cytarabine was revealed in the presence of plasmin.