Fernando C. Giacomelli

Formation of Catalytic Silver Nanoparticles Supported on Branched Polyethyleneimine Derivatives

A new and straightforward method for screening highly catalytically active silver nanoparticle-polymer composites derived from branched polyethyleneimine (PEI) is reported. The one-step systematic derivatization of the PEI scaffold with alkyl (butyl or octyl) and ethanolic groups led to a structural diversity correlated to the stabilization of silver nanoparticles and catalysis. Analysis of PEI derivative libraries identified a silver nanoparticle-polymer composite that was able to efficiently catalyze the p-nitrophenol reduction by NaBH(4) in water with a rate constant normalized to the surface area of the nanoparticles per unit volume (k(1)) of 0.57 s(-1) m(-2) L. Carried out in the presence of excess NaBH(4), the catalytic reaction was observed to follow pseudo-first-order kinetics and the apparent rate constant was linearly dependent on the total surface area of the silver nanoparticles (Ag-NPs), indicating that catalysis takes place on the surface of the nanoparticles. All reaction kinetics presented induction periods, which were dependent on the concentration of substrates, the total surface of the nanoparticles, and the polymer composition. All data indicated that this induction time is related to the resistance to substrate diffusion through the polymer support. Hydrophobic effects are also assumed to play an important role in the catalysis, through an increase in the local substrate concentration.

Combination chemotherapy using core-shell nanoparticles through the self-assembly of HPMA-based copolymers and degradable polyester.

The preparation of core-shell polymeric nanoparticles simultaneously loaded with docetaxel (DTXL) and doxorubicin (DOX) is reported herein. The self-assembly of the aliphatic biodegradable copolyester PBS/PBDL (poly(butylene succinate-co-butylene dilinoleate)) and HPMA-based copolymers (N-(2-hydroxypropyl)methacrylamide-based copolymers) hydrophobically modified by the incorporation of cholesterol led to the formation of narrow-size-distributed (PDI<0.10) sub-200-nm polymeric nanoparticles suitable for passive tumor-targeting drug delivery based on the size-dependent EPR (enhanced permeability and retention) effect. The PHPMA provided to the self-assembled nanoparticle stability against aggregation as evaluated in vitro. The highly hydrophobic drug docetaxel (DTXL) was physically entrapped within the PBS/PBDL copolyester core and the hydrophilic drug doxorubicin hydrochloride (DOX·HCl) was chemically conjugated to the reactive PHPMA copolymer shell via hydrazone bonding that allowed its pH-sensitive release. This strategy enabled the combination chemotherapy by the simultaneous DOX and DTXL drug delivery. The structure of the nanoparticles was characterized in detail using static (SLS), dynamic (DLS) and electrophoretic (ELS) light scattering besides transmission electron microscopy (TEM). The use of nanoparticles simultaneously loaded with DTXL and DOX provided a more efficient suppression of tumor-cell growth in mice bearing EL-4 T cell lymphoma when compared to the effect of nanoparticles loaded with either DTXL or DOX separately. Additionally, the obtained self-assembled nanoparticles enable further development of targeting strategies based on the use of multiple ligands attached to an HPMA copolymer on the particle surface for simultaneous passive and active targeting and different combination therapies.

pH-triggered block copolymer micelles based on a pH-responsive PDPA (poly[2-(diisopropylamino)ethyl methacrylate]) inner core and a PEO (poly(ethylene oxide)) outer shell as a potential tool for the cancer therapy

The potential of a novel pH-triggered block copolymer as a promising drug delivery platform for the cancer therapy has been explored. The block copolymer poly(ethylene oxide)-b-poly(glycerol monomethacrylate)-b-poly[2-(diisopropylamino)ethyl methacrylate] herein referred to as PEO113-b-PG2MA30-b-PDPA50 upon dissolution in ethanol followed by single-step nanoprecipitation in phosphate buffered saline (PBS) self-assembled into highly regular spherical micelles whose structure was characterized in detail by static (SLS), dynamic (DLS) and electrophoretic (ELS) light scattering, small angle X-ray scattering (SAXS), fluorescence spectroscopy and transmission electron microscopy (TEM). The micellar size (2RH = 42 nm) and micellar molecular weight (Mw(micelles) > 106 kDa) were found to be in the range to avoid renal clearance providing a long blood circulation time. Their size is below the cut-off size of the leaky pathological vasculature (DH < 200 nm), making them candidates for the use in cancer therapy based on the EPR effect. The pH-responsive PDPA core could be loaded with the poorly water-soluble anti-cancer drug paclitaxel (PTX) with encapsulation efficiency ∼70% and drug loading content ∼7% wdrug/wpolymer. The pKa of the diisopropylamino group of the PDPA block was determined as pKa = 6.8 in the simulated physiological condition, which is remarkably close to the pH microenvironment of tumoral cells. The release experiments evidenced that approximately 90% of the encapsulated PTX was sustained at the PDPA micellar core within the first 9 h at pH 7.4 whilst only 18 h were required for complete drug release at pH 5.0. These results suggest that the micellar dissociation might be triggered at the slightly acid tumoral extracellular environments (pH < pKa(PDPA)). The nanostructures were further placed in contact with human plasma or human serum albumin (HSA) diluted in PBS. The DLS experiments revealed that the micelles are especially stable for up to at least 48 h in such conditions, attesting the possibly long blood circulation time of the nanoparticles at serum environments which is a pre-requisite for the drug delivery applications. The cell viability experiments demonstrated that the drug-free block copolymer micelles are non-toxic and the number of viable cells is always greater than 85% compared to the survival number of a control group.

Aggregation behavior of a new series of ABA triblock copolymers bearing short outer A blocks in B-selective solvent: from free chains to bridged micelles.

A combination of dynamic (DLS) and static (SLS) light scattering measurements was employed to study the self-assembly behavior of a new series of triblock copolymers bearing poly[5-(N,N-diethylamino isoprene)] (PAI) short outer blocks and polystyrene (PS) as the major middle block. Previously, it was verified that PAI outer blocks can be quaternized leading the formation of crew-cut aggregates in water (Riegel, I. C.; Eisenberg, A.; Petzhold, C. L.; Samios, D. Langmuir 2002, 18, 3358). Herein, we focus on the copolymers ability in the nonquaternized version to undergo self-aggregation in dimethylformamide (DMF), a selective solvent for the middle block. Light scattering measurements showed that formation of well-defined flowerlike micelles is likely to occur. Aggregates with a relatively narrow distribution, small average size, and number of aggregation ranging from 21 to 39 chains/micelle were experimentally observed. The results also suggested that approximately 5-6 polymeric units per each short outer block are needed to induce aggregation. The middle block length governs the size of the micelles and influences the number of aggregation of the resultant particles as well. Furthermore, when the polystyrene middle block was particularly long (degree of polymerization DP > 600), dynamic and static light scattering measurements suggested the formation of bridged micelles in an open structure in concentrations as low as 15 mg mL-1.

Self-Assembly of Amphiphilic Glycoconjugates into Lectin-Adhesive Nanoparticles

This work describes the synthesis and self-assembly of carbohydrate-clicked rod-coil amphiphilic systems. Copper-catalyzed Huisgen cycloaddition was efficiently employed to functionalize the hydrophilic extremity of PEG-b-tetra(p-phenylene) conjugates by lactose and N-acetyl-glucosamine ligands. The resulting amphiphilic systems spontaneously self-assembled into nanoparticles when dissolved in aqueous media, as evidenced by dynamic light scattering (DLS), transmission electron microscopy (TEM), and small-angle X-ray scattering (SAXS). The formation of highly monodisperse micelles having a mean diameter of 10 nm was observed for systems containing a PEG 900 core, and a decrease in the hydrophilic moiety (PEG 600) led to the formation of vesicles with a broader size distribution. The presence of carbohydrate residues on the surfaces of the micelles and their ability to establish specific interactions with wheat germ agglutinin (WGA) and peanut agglutinin (PNA) were further highlighted by light-scattering measurements, thus confirming the attractive applications of such sugar micelles in biosensor devices.

Novel “soft” biodegradable nanoparticles prepared from aliphatic based monomers as a potential drug delivery system

The search for new biomaterials intended for biomedical applications has considerably intensified in recent years. Herein, the synthesis and characterization of a new aliphatic biodegradable copolyester named PBS/PBDL (poly(butylene succinate-co-butylene dilinoleate)) is reported. Surfactant-free, narrowly distributed, nanosized spherical particles (RH < 60 nm) have been produced from the biodegradable material by applying a single-step nanoprecipitation protocol. Their structure was characterized in detail by employing a variety of scattering techniques and transmission electron microscopy (TEM). Combined SLS and DLS measurements suggested that the nanoparticles comprise a porous core conferring a non-compact characteristic. Their porosity enables water to be entrapped which is responsible for their pronounced stability and relatively fast degradation as followed by size exclusion chromatography (SEC). The polymeric nanoparticles could be loaded with the hydrophobic model drug paclitaxel (PTX) with an encapsulation efficiency of ∼95% and drug loading content of ∼6–7% wdrug/wpolymer. The drug release was followed by HPLC and scattering measurements (DLS, SLS and SAXS). The drug encapsulation and release modifies the inner structure of the nanoparticles, which holds a large amount of entrapped water in the drug-free condition. PTX encapsulation leads to replacement of the entrapped water by the hydrophobic model drug and to shrinking of the nanoparticles, probably due to favorable drug–polymer hydrophobic interactions. Cell viability experiments demonstrated that the nanoparticles are biocompatible and non-toxic, making them potentially useful for applications in nanomedicine.

Self-assembly of biodegradable copolyester and reactive HPMA-based polymers into nanoparticles as an alternative stealth drug delivery system

The surface modification of nanoparticles by physically anchoring hydrophilic biocompatible polymers is a simple and commercially attractive strategy to produce stealth drug delivery nanocarriers. Herein, we report the preparation, characterization and preliminary evaluation of the biological behaviour of polymeric nanoparticles (NPs) comprising a biodegradable poly(butylene succinate-co-butylene dilinoleate) – PBS/PBDL – copolyester and a non-immunogenic and non-toxic hydrophilic N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Narrowly distributed sub-100 nm polymeric nanoparticles with stealth properties were successfully prepared by using a combination of interfacial nanoprecipitation and self-assembly. The assemblies were characterized by using complementary scattering techniques and cryo-transmission electron microscopy. The dimension of the NPs was found to be in the proper range to avoid fast renal clearance (DH > 10 nm) and still below the cut-off size of the leaky pathological microvasculature of hypervascular tumours (DH < 200 nm), thus making them candidates for application in cancer therapy based on the EPR effect. The presence of PHPMA copolymer exposed at the surface of the nanoparticles was confirmed by scattering measurements. The stealth property of the biocompatible and biodegradable NPs is responsible for their remarkable in vitro stability monitored in a simulated physiological environment and increased stability in concentrated NaCl solutions compared to uncoated PBS/PBDL nanoparticles, making them an alternative to PEG-shielded particles. Furthermore, a reproducible, efficient and satisfactory physical entrapment of the antitumoral drug doxorubicin (DOX) was achieved (∼5.0% wdrug/wNPs). The controlled DOX release is pH-dependent and faster under slightly acidic conditions and the cell viability experiments demonstrated that the drug-free NPs are non-toxic, whereas the DOX-loaded NPs exert in vitro cytostatic efficacy on EL4 T cell lymphoma.

Direct synthesis of coated gold nanoparticles mediated by polymers with amino groups.

The single-step/single-phase synthesis of hybrid organic-inorganic core-shell gold nanoparticles (AuNPs), facilitated by amino-functionalized amphiphilic block copolymers that simultaneously play the roles of reductant and stabilizer, was investigated in this study. Experiments were devised with emphasis on the pH-responsive poly(ethylene oxide)-b-poly(2,3-dihydroxypropyl methacrylate)-b-poly[2-(diisopropylamino)ethyl methacrylate] triblock copolymer, which allows direct chemical cross-linking of the micellar structures to be performed. The polymer structure-reactivity relationship associated with the AuNP formation was established using a set of six structurally related macromolecules. AuNP formation was dependent on the aqueous dissociation equilibrium involving tertiary amino groups, the Au(III) speciation, and electrochemical redox potentials. The effects of these parameters on the synthesis of AuNPs change as the solution pH is increased from pH 3.3 (molecularly dissolved polymer chains; no AuNP formation) to 6.8 or higher (polymer chains self-assembled into spherical micelles; stable gold sols are produced), and Au(III) reduction potentials shift toward the cathodic region while the oxidation potential of deprotonated amino groups decreases. Sigmoidal nanoparticle growth kinetics was observed in all cases after a characteristic induction period. Stable, well-defined, uniform polymer-coated gold colloids with localized surface plasmon resonance centered at 53 0nm can be conveniently produced in one-pot, two-reactant, no work-up reactions when the stoichiometry is [N]/[Au]=3.5-25.0.

Light scattering evidence of selective protein fouling on biocompatible block copolymer micelles

Selective protein fouling on block copolymer micelles with well-known potential for tumour-targeting drug delivery was evidenced by using dynamic light scattering measurements. The stability and interaction of block copolymer micelles with model proteins (BSA, IgG, lysozyme and CytC) is reported for systems featuring a hydrophobic (poly[2-(diisopropylamino)-ethyl methacrylate]) (PDPA) core and hydrophilic coronas comprising poly(ethylene oxide)/poly(glycerol monomethacrylate) (PEO-b-PG2MA) or poly[2-(methacryloyloxy)ethyl phosphorylcholine] (PMPC). The results revealed that protein size and hydrophilic chain density play important roles in the observed interactions. The PEO(113)-b-PG2MA(30)-b-PDPA(50) nanoparticles are stable and protein adsorption is prevented at all investigated protein environments. The successful protein-repellent characteristic of these nanoparticles is attributed to a high hydrophilic surface chain density (>0.1 chains per nm(2)) and to the length of the hydrophilic chains. On the other hand, although PMPC also has protein-repellent characteristics, the low surface chain density of the hydrophilic shell is supposed to enable interactions with small proteins. The PMPC(40)-b-PDPA(70) micelles are stable in BSA and IgG environments due to weak repulsion forces between PMPC and the proteins, to the hydration layer, and particularly to a size-effect where the large BSA (R(H) = 4.2 nm) and IgG (R(H) = 7.0 nm) do not easily diffuse within the PMPC shell. Conversely, a clear interaction was observed with the 2.1 nm radius lysozyme. The lysozyme protein can diffuse within the PMPC micellar shell towards the PDPA hydrophobic core in a process favored by its smaller size and the low hydrophilic PMPC surface chain density (∼0.049 chains per nm(2)) as compared to PEO-b-PG2MA (∼0.110 chains per nm(2)). The same behavior was not evidenced with the 2.3 nm radius positively charged CytC, probably due to its higher surface hydrophilicity and the consequent chemical incompatibility with PDPA.

Self-assembled carbohydrate-based micelles for lectin targeting

Biocompatible low-polydispersity micelles designed for lectin targeting have been prepared by spontaneous self-assembly in water of macromolecular glycosylated amphiphiles. Propargyl-β-lactoside and N-acetyl-β-D-glucosaminide were conjugated by copper-catalyzed Huisgen cycloaddition to azide-terminated PEG 900 stearate. Upon dissolution in water, the resulting amphiphiles immediately self-assemble into highly regular micelles having a mean diameter of 10 nm. Dynamic Light Scattering (DLS), Transmission Electron Microscopy (TEM) and Small-Angle X-ray Scattering (SAXS) were used to investigate the structure of the self-assembled saccharidic amphiphiles micelles. The presence of the carbohydrate epitopes on the surface of the micelles and their bioavailability for lectin targeting were also demonstrated by light scattering measurements. Specific interaction of the GlcNac and Lac residues with Wheat Germ Agglutinin (WGA) and Peanut Agglutinin (PNA) respectively, unveils potential applications of such carbohydrate-derived surfactants as simple and site-specific vectorization systems for drug delivery.