Poly(amidoamine)-Cholesterol Conjugate Nanoparticles Obtained by Electrospraying as Novel Tamoxifen Delivery System.

A new poly(amidoamine)-cholesterol (PAA-cholesterol) conjugate was synthesized, characterized and used to produce nanoparticles by the electrospraying technique. The electrospraying is a method of liquid atomization that consists in the dispersion of a solution into small charged droplets by an electric field. Tuning the electrospraying process parameters spherical PAA-chol nanoparticles formed. The PAA-cholesterol nanoparticles showed sizes lower than 500 nm and spherical shape. The drug incorporation capacity was investigated using tamoxifen, a lipophilic anticancer drug, as model drug. The incorporation of the tamoxifen did not affect the shape and sizes of nanoparticles showing a drug loading of 40%. Tamoxifen-loaded nanoparticles exhibited a higher dose-dependent cytotoxicity than free tamoxifen, while blank nanoparticles did not show any cytotoxic effect at the same concentrations. The electrospray technique might be proposed to produce tamoxifen-loaded PAA-chol nanoparticle in powder form without any excipient in a single step.

Structural characterisation of poly(amidoamine) networks via high-resolution magic angle spinning NMR.

A comprehensive structural characterisation of cross-linked insoluble poly(amidoamine) (PAA) networks was performed by high-resolution magic angle spinning (HRMAS) NMR spectroscopy. Model samples with 20%, 40% and 80% cross-linking degrees were prepared and the best conditions to obtain high-resolution spectra in the gel phase determined. Whereas the samples with 20% and 40% cross-linking degrees could be exhaustively resolved and described, the sample with 80% cross-linking degree could not be characterised by this technique owing to insufficient mobility of the polymer segments. Even with this limitation, the method developed in this study can be reasonably considered as a general one, which enables exhaustive characterisation of cross-linked PAA networks of biomedical interest.

Micro- and nanoscale modification of poly(2-hydroxyethyl methacrylate) hydrogels by AFM lithography and nanoparticle incorporation.

Poly(2-hydroxyethyl methacrylate) (PHEMA) hydrogels are widely used as biomaterials. Due to their unique combination of biocompatibility and good mechanical properties, they have potential as scaffolds for tissue engineering applications. To this purpose, topographic and chemical patterning at the nano- to the mesoscale is crucial in order to favor and to characterize cell adhesion and proliferation. Here we report the characterization of as-prepared and patterned PHEMA hydrogels, produced by conventional radical polymerization in water and dimethylformamide. We have obtained chemical and morphological micro- and nanoscale patterning by atomic force microscopy based lithography. We also demonstrate that it is possible to incorporate carbon nanoparticles in the hydrogel matrix by supersonic cluster beam deposition.

Evidence of aggregation in dilute solution of amphoteric poly(amido-amine)s by size exclusion chromatography.

Evidence of aggregation of amphoteric linear poly(amido-amine)s (PAAs) was proved using a multi-angle laser light scattering detector on-line to a size exclusion chromatography (SEC) system. As a rule the PAAs chemical structure, with the presence of charged groups that are both anionic and cationic, easily generates aggregation and non-steric fractionation. A non-amphoteric, non-aggregate PAAs polymer with an elution pattern close to ideal SEC was also obtained and characterized for comparison. The influence of PAAs synthesis conditions on the extent of aggregation was also studied.

Understanding the mechanism of action of poly(amidoamine)s as endosomolytic polymers: correlation of physicochemical and biological properties.

Bioresponsive poly(amidoamine)s (PAA)s are currently under development as endosomolytic polymers for intracellular delivery of proteins and genes. Here for the first time, small-angle neutron scattering (SANS) is used to systematically investigate the pH-dependent conformational change of an endosomolytic polymer, the PAA ISA 23. The radius of gyration of the ISA23 was determined as a function of pH and counterion, the aim being to correlate changes in polymer conformation with membrane activity assessed using a rat red blood cell haemolysis assay. With decreasing pH, the ISA23 radius of gyration increased to a maximum (R(g) approximately 80 A) around pH = 3, before subsequently decreasing once more. At high pH and therefore high ionic strengths, the polymer is negatively charged and adopts a rather compact structure (R(g) approximately 20 A), presumably with the dissociated carboxylic groups on the exterior of the polymer coil. At low pH, the coil again collapses (R(g) < 20 A), presumably due to the effects of the high ionic strength. It is concluded that the nature of the salt form has no direct bearing on the size of the polymer coil, but it does indirectly determine the prevailing pH and, hence, polymer conformation. Pulsed-gradient spin-echo NMR measurements were in good agreement with the SANS estimates of the radius of gyration, although ISA23 polydispersity does complicate the data interpretation/comparison. These results support the proposed mode of action of PAAs, namely a coil expansion on passing from a neutral pH (extracellular) to an acidic pH (endosomal and lysosomal) environments. The results do, however, suggest that the charge on the polymer shows a closer correlation with the haemolysis activity rather than the polymer conformation.

Poly(amidoamine)-mediated intracytoplasmic delivery of ricin A-chain and gelonin.

Poly(amidoamine)s (PAAs) are water-soluble synthetic polymers designed to be biodegradable and biocompatible. Moreover, they display membrane disruptive properties in response to a decrease in pH. This attribute confers PAAs with endosomolytic properties in vitro and in vivo. A model system was developed to quantify their ability to promote the endosomal escape of macromolecules that may be interesting as therapeutic agents. Here, two PAAs (ISA 1 and 4) were incubated with B16F10 cells in vitro together with two non-permeant toxins: either ricin A-chain (RTA) or gelonin. The relatively non-toxic PAAs ISA 1 and 4 (IC50>1.5 mg/ml) restored activity to the inherently inert toxins. The IC50 values for the ISA 1/RTA and ISA 1/gelonin combinations were 0.65+/-0.05 and 0.55+/-0.12 mg/ml, respectively. Similarly, when ISA 4 was incubated with a non-toxic combination of RTA and gelonin the IC50 value decreased to 0.57+/-0.03 and 0.43+/-0.26 mg/ml, respectively. In contrast, the neutral polymer dextran and the PAA ISA 22 were unable to mediate this effect. These observations suggest that specific PAA-toxin combinations warrant further development as novel therapeutics.

Poly(amidoamine)s as potential nonviral vectors: ability to form interpolyelectrolyte complexes and to mediate transfection in vitro.

Poly(amidoamine)s (PAAs) are water-soluble polymers that display pH-dependent membrane activity. PAAs have the potential to act as a synthetic alternative to fusogenic peptides and thus promote endosomal escape. The purpose of this study was to investigate for the first time whether PAA have the ability to complex DNA, protect it from nuclease degradation and to promote transfection in vitro. PAAs ISA 1 (Mn 6900) and ISA 23 (Mn 10,500) and their 2-phenylethylamine containing analogues ISA 4 and ISA 22 (Mn approximately 8000) were studied. All PAAs retarded the electrophoretic mobility of lambda Hind III DNA demonstrating interpolyelectrolyte complex (IPEC) formation and toroids of 80-150 nm in diameter (10:1 polymer excess) were visible using TEM. DNase II inhibition was observed. At a polymer:DNA ratio of 10:1, this was ISA 1(89.6 +/- 6.1%), ISA 4 (92.2 +/- 11.2%), ISA 22 (69.4 +/- 3.7%), and ISA 23 (58.0 +/- 10.0%). PAAs demonstrated the ability to mediate pSV beta-galactosidase transfection of HepG2 cells. At a vector:DNA mass ratio of 5:1, ISA 23 showed equivalent transfection ability compared with polyethylenimine and LipofectIN and was more effective than LipofectACE. These properties suggest that PAAs warrant further development as endosomolytic vectors.

Preparation and characterisation of rose Bengal-loaded surface-modified albumin nanoparticles.

Surface-modified albumin nanoparticles were prepared from two poly(ethylene glycol)-human serum albumin conjugates: poly(thioetheramido acid)-poly(ethylene glycol) copolymer-grafted HSA (HSA-PTAAC-PEG) and methoxy poly(ethylene glycol)-grafted HSA (HSA-mPEG). Rose bengal (RB) was used as a model drug for encapsulation into the nanoparticles either during the particle production or by adsorption post particle preparation. The drug incorporation and release was affected by the different production methods and the different polymer compositions. When RB was loaded in HSA and HSA/HSA-PTAAC-PEG nanoparticles, up to 5% (w/w) drug content was achieved. The drug loading in HSA-mPEG nanoparticles was much lower and the results from the microcalorimetry study indicated that the low loading efficiency was due to less drug-protein binding sites available in the HSA-mPEG molecule as compared to the HSA molecule. The release of RB from the albumin nanoparticles was very slow in PBS and dramatically accelerated in the presence of trypsin. Compared with unmodified nanoparticles, the slower release of RB from the surface-modified HSA nanoparticles in the presence of the enzyme suggested that the existence of a steric hydrophilic barrier on the surface of the nanoparticles made digestion of the nanoparticles more difficult.

The desorption process of macromolecules adsorbed on interfaces: the force spectroscopy approach.

They're flexible and sticky: While investigations of the very stiff DNA molecular conformation on surfaces have been made, the equivalent for more typical macromolecules is complicated by their shorter persistance length. A gentle detachment study with a polymer bound to an SFM tip allows the forces between the polymer and surface to be probed; the detachement force required depends on the surface conformation, whether only a small loop or a long tail must be peeled from the surface, as shown by the cartoon.

PAcM-AN: poly (N-acryloylmorpholine)-conjugated antisense oligonucleotides.

A new amphiphilic, high-molecular weight poly (N-acryloylmorpholine) (PAcM) polymer has been used to be linked to oligonucleotide chains through a liquid-phase stepwise synthesis. This new conjugate has been investigated for its melting property, nuclease stability and capacity to elicit RNase H activity. Its antisense activity against an HIV-1 target has been also evaluated.

Therapeutic proteins: a comparison of chemical and biological properties of uricase conjugated to linear or branched poly(ethylene glycol) and poly(N-acryloylmorpholine).

Uricase from Bacillus fastidiosus (UC) was covalently linked to linear PEG (PEG-1) (Mw 5 kDa), branched PEG (PEG-2) (Mw 10 kDa) and to poly(N-acryloylmorpholine) (PAcM) (Mw 6 kDa). The conjugation of UC with linear PEG and PAcM was accompanied by complete loss of enzymatic activity but, if uric acid as site protecting agent was included in the reaction mixture, the conjugate protein retained enzymatic activity. On the other hand, the modification with PEG-2 gave a conjugate that also maintained enzymatic activity in the absence of any active site protection. This behaviour must be related to hindrance of the branched polymer in reaching the enzyme active site. The UC conjugates exhibited increased resistance to proteolytic digestion while minor variations in the inhibitory constant, optimal pH, heat stability, affinity for substrate, were observed. Pharmacokinetic investigations in mice demonstrated increased residence time in blood for all the conjugates as compared with native uricase. Uricase conjugated with linear PEG was longer lasting in blood UC derivative, followed by branched PEG and the PAcM conjugates. Unconjugated uricase was rapidly removed from circulation. All these data are in favour of the use of the less known amphiphilic polymer PAcM as an alternative to PEGs in modification of enzymes devised for therapeutic applications.

Poly(amidoamine)s as potential endosomolytic polymers: evaluation in vitro and body distribution in normal and tumour-bearing animals.

Fusogenic peptides derived from viral coat proteins cause perturbation of the endosomal membrane and are often used to improve the transfection efficiency of non-viral vectors in vitro. However, fusogenic peptides have limited potential for use in vivo due to their inherent immunogenicity. Totally synthetic polymers that are endosomolytic should circumvent this problem and could be useful as components of non-viral delivery systems as long as they do not immediately localise in the liver after intravenous (i.v.) injection. Linear poly(amidoamine) polymers (PAAs) having amido- and tertiary amino-groups along the main polymer undergo pH-dependent conformational change and thus provide an ideal opportunity for design of polymers that display membrane activity at low pH. Here we describe four PAAs, ISA 1 (Mn = 6900 Da) and ISA 23 (Mn = 10,500 Da) and their analogues ISA 4 and ISA 22 (Mn approximately 8000 Da) containing approximately 1 mol% 2-p-hydroxyphenyl ethylamine to allow radioiodination and thus monitoring of their biodistribution. In vitro cytotoxicity was assessed by MTT assay after incubation of PAAs with B16F10 and Mewo cell lines. The IC50 values observed for all PAAs were > 2 mg/mL in comparison with poly(L-lysine) which displayed an IC50 in the range 0.01-0.1 mg/mL. At pH 7.4 none of the PAAs studied was haemolytic at 1 h at concentrations below 3 mg/mL. PAAs were subsequently incubated with rat red blood cells for 24 h (1 mg/mL) at different pHs. In contrast to poly(L-lysine) which was haemolytic at pH 7.4, 6.5 and 5.5, none of the PAAs was lytic at pH 7.4, but they became membrane active at lower pH (approximately 45% for ISA 4, 50% for ISA 22 and 90% for ISA 23). These observations were substantiated by SEM and confirm the pH-dependence of membrane activity. After i.v. injection to rats 125I-labelled ISA 4 was immediately taken up by the liver (> 80% recovered dose at 1 h) whereas 125I-labelled ISA 22 was not (liver uptake was < 10% recovered dose at 5 h). Furthermore, biodistribution studies in mice bearing subcutaneous B16F10 melanoma showed that 125I-labelled ISA 22 was still accumulating in tumour tissue after 5 h (2.5% dose/g). PAAs have potential as endosomolytic agents and quantitation of the endosome to cytoplasm transfer is warranted after i.v. administration.

Preparation of surface-modified albumin nanospheres.

Surface-modified human serum albumin (HSA) nanospheres with a size of around 100 nm in diameter were prepared from poly(amidoamine)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PAA-PEG) and poly(thioetheramido acid)-poly(ethylene glycol) copolymer grafted human serum albumin (HSA-PTAAC-PEG). The nanospheres were produced using a pH-coacervation method and cross-linked with glutaraldehyde. The cross-linking efficiency was affected by the type of albumin conjugate used. The zeta potential of the surface-modified nanospheres was significantly lower than that of unmodified particles. The existence of a hydrated steric barrier surrounding the nanospheres was confirmed by electrolyte- and pH-induced flocculation tests. The surface-modified nanospheres showed a reduced plasma protein adsorption on the particle surface compared with unmodified particles.

Synthesis and pharmacokinetic behaviour of ester derivatives of 4-isobutylphenyl-2-propionic acid (Ibuprofen) with end-hydroxylated poly(N-vinyl pyrrolidinone) and poly(N-acryloyl morpholine) oligomers.

Four derivatives of 4-isobutylphenyl-2-propionic acid (Ibuprofen), in which the drug was bound by ester linkages to poly(ethylene glycols) (PEG 2000-I), monomethoxy poly(ethylene glycols) (PEG 1900-I), poly(N-vinyl pyrrolidinone) (PVP-I) and poly(N-acryloyl morpholine) (PACM-I), all having approximatively the same number average molecular weight (Mn congruent equal to 2000), were prepared and tested for their pharmacokinetic properties after oral administration. It was found that the two end-hydroxylated amphiphilic oligomers of polyvinylic structure, PACM and PVP, whose physico-chemical properties are comparable to those of PEGs especially as regards solvent affinity, have in principle a similar potential as promoieties for preparing oligomeric prodrugs.

Degradation behaviour of block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments.

The degradation behaviour of a new class of multi-block copolymers containing poly(D,L-lactic-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) segments was studied under conditions mimicking those found in biological fluids. The dissolution times of the new PLGA-PEG multi-block copolymers mainly depend on the length of the PEG segments present in their structure, i.e., on their PEG content on a weight/weight basis. At higher PEG contents, partially degraded PLGA segments are brought in solution by attached PEG segments. In all cases, the dissolution rates decrease by increasing the total surface area of the specimens tested.

Synthesis and properties of novel block copolymers containing poly(lactic-glycolic acid) and poly(ethyleneglycol) segments.

A synthetic process for obtaining high-molecular-weight block copolymers containing poly(lactic-glycolic acid) and poly(ethylene glycol) segments has been established. This process involves the reaction of poly(ethylene glycols) with phosgene, followed by polycondensation of the resulting alpha, omega-bis(chloroformates) with poly(lactic-glycolic acid) oligomers. The copolymers have been characterized for their molecular weight, solubility properties, water absorption and preliminarily thermal behaviour. All evidence points to the conclusion that the process described is a general one, enabling biodegradable polymers to be obtained tailor-made according to specific requirements.

New synthetic amphiphilic polymers for steric protection of liposomes in vivo.

Carboxy group-terminated synthetic polymers--branched poly(ethylene glycol), poly(acryloylmorpholine), and poly(vinylpyrrolidone)--were made amphiphilic by derivatization with phosphatidyl ethanolamine via the terminal carboxy group and then incorporated into lecithin-cholesterol liposomes prepared by the detergent dialysis method. Following the biodistribution of liposomes in mice, all three polymers were shown to be effective steric protectors for liposomes and were able to sharply increase liposome circulation times in a concentration-dependent manner. The accumulation of liposomes in the liver decreases. The effects observed are similar to those found for liposomes modified with linear poly(ethylene glycol). At low polymer concentration, amphiphilic branched poly(ethylene glycol) seems to be the most effective liposome protector, most probably, because at the same molar content of anchoring groups, each attachment point carries two polymeric chains and doubles the quantity of liposome-grafted polymer comparing to linear poly(ethylene glycol).

Degradation behaviour of ionic stepwise polyaddition polymers of medical interest.

The aim of this presentation is to review some of our recent work mostly on poly(amidoamine)s (PAAs) and some other families of polymers structurally related to PAAs of medical interest. PAAs are obtained by stepwise polyaddition of primary monoamines, or bis secondary amines, to bisacrylamides. There are several other ter-amino polymers structurally related to PAAs, such poly(amido phosphine)s (PAPs), poly(ester-amine)s (PEAs), poly(ketone-amine)s (PKAs), poly(amidothioeteramine)s (PATAs) poly(esterthioether amine)s (PTEAs), and poly(sulphone thioetheramine)s (PSTAs). Most of the PAAs exhibit heparin complexing ability. PAAs are also being considered as soluble carriers for delivering anti-cancer drugs. Some of these polymers have been studied as antimicrobial agents. PAAs with different structures degrade at different rates under physiological conditions. The degradation rate is also strongly influenced by pH. The quaternarized PATAs and PTEAs are reasonably stable over a period of some days, but ultimately degrade to oligomeric products, while the quaternized PAAs do rapidly degrade.

Recent results on functional polymers and macromonomers of interest as biomaterials or for biomaterial modification.

Different families of functionalized polymers with potential as biomaterials, or for biomaterial modification, have been investigated. In particular, degradation studies have been performed on poly(amidoamines), a family of polymers obtained by polyaddition of amines to bisacrylamides, and endowed with heparin-complexing ability. Some new poly(amidoamines) with more resistance towards hydrolytic degradation than traditional ones have been discovered. Other ter-amino polymers deriving from the polyaddition of ter-amino functionalized bis-thiols to bis-acrylic esters, or other activated unsaturated compounds, have been studied. Their quaternarization products have been proven, in a parallel work, to act as powerful antimicrobial agents. By performing in situ the polyaddition reaction, semi-interpenetrated networks based on silicone rubber and the same polymers have been prepared. Finally, end-functionalized amphiphilic oligomers have been prepared by radical polymerization techniques, and their use for enzyme modification considered.

A comparison between the hemolytic and antibacterial activities of new quaternary ammonium polymers.

New quaternary ammonium polymers, which in a previous work had shown relevant antibacterial properties, have been investigated as regards to their hemolytic activity (HA) in comparison with a low molecular weight commercial antibacterial agent, Steramine G (SG). All polymers exhibit negligible, or at most modest, HA at dosages and contact times at which SG is strongly hemolytic.