Francesca Mastrotto

Tailored PEG for rh-G-CSF analogue site-specific conjugation.

A new end-tailored monomethoxypoly(ethylene glycol) (PEG) for site-directed protein conjugation was synthesized according to a three-step procedure: (1) linear 20 kDa PEG-NH(2) was conjugated to 12-(Boc-amino)dodecanoic acid; (2) PEG-NHCO(CH(2))(11)-Boc was deprotected by TFA treatment; (3) PEG-NHCO(CH(2))(11)-NH(2) was conjugated to 6-maleimidohexanoic acid to yield PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal (PEG-C(18)-Mal). The chemical intermediates as well as the final product were purified by solvent precipitation/extraction and characterized by (1)H NMR spectroscopy and colorimetric analysis. The synthesis procedure yielded over 90% activated product [PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal/PEG-NH(2) molar ratio, %]. Both PEG-C(18)-Mal and the commercial maleimido activated 20 kDa linear PEG (PEG-Mal) were used for conjugation to (17)Cys of recombinant human granulocyte colony stimulating factor (rh-G-CSF). Under denaturing conditions, at pH 7.0, both activated PEGs yielded over 90% protein conjugation. Under native conditions, about 55% and 7% PEGylated protein were obtained with PEG-C(18)-Mal and PEG-Mal, respectively. Circular dichroism analysis showed that the PEGylation does not induce detectable alteration of the protein secondary structure. On the other hand, the PEGylation conditions were found to affect significantly the protein stability. The derivatives obtained either with the two polymers by unfolding/refolding process or with PEG-Mal under native conditions displayed rapid aggregation with half-life ranging from 30 to 90 min. The derivative obtained with PEG-NHCO-(CH(2))(11)-NHCO(CH(2))(5)-Mal in the absence of guanidinium chloride displayed remarkably higher stability with aggregation half-life of about 60 h.

Self-assembling nanocomposites for protein delivery: supramolecular interactions between PEG-cholane and rh-G-CSF.

PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane self-assembling polymers have been synthesised by the end-functionalisation of 5, 10 and 20 kDa linear amino-terminating monomethoxy-poly(ethylene glycol) (PEG-NH(2)) with 5β-cholanic acid. Spectroscopic studies and isothermal titration calorimetry showed that the CMC of the PEG-cholane derivatives increased from 23.5 ± 1.8 to 60.2 ± 2.4 μM as the PEG molecular weight increased. Similarly, light scattering analysis showed that the micelle size increased from 15.8 ± 4.9 to 23.2 ± 11.1 nm with the PEG molecular weight. Gel permeation studies showed that the polymer bioconjugates associate with recombinant human granulocyte colony stimulating factor (rh-G-CSF) to form supramolecular nanocomposites according to multi-modal association profiles. The protein loadings obtained with PEG(5 kDa)-cholane, PEG(10 kDa)-cholane and PEG(20 kDa)-cholane were 7.4 ± 1.1, 2.7 ± 0.3 and 2.1 ± 0.4% (protein/polymer, w/w %), respectively. Scatchard and Klotz analyses showed that the protein/polymer affinity constant increased and that the number of PEG-cholane molecules associated to rh-G-CSF decreased as the PEG molecular weight increased. Isothermal titration calorimetry confirmed the protein/polymer multi-modal association. Circular dichroism analyses showed that the polymer association alters the secondary structure of the protein. Nevertheless, in vitro studies performed with NFS-60 cells showed that the polymer interaction does not impair the biological activity of the cytokine. In vivo studies performed by intravenous and subcutaneous administrations of rh-G-CSF to rats showed that the association with PEG(5 kDa)-cholane prolongs the body exposure of the protein. After subcutaneous administration, the protein t(max) values obtained with rh-G-CSF and 1:14 and 1:21 rh-G-CSF/PEG(5 kDa)-cholane (w/w ratio) nanocomplexes were 2, 8 and 24h, respectively. The 1:21 (w/w) rh-G-CSF/PEG(5kDa)-cholane formulation resulted in 149% relative bioavailability, and the pharmacokinetic behaviour was similar to that obtained with an equivalent protein dose of rh-G-CSF chemically conjugated with one linear 20-kDa PEG. A single administration of a 1.5mg/kg dose of a 1:21 (w/w) rh-G-CSF/PEG(5 kDa)-cholane formulation induced a high production of white blood cells for 96 h.

pH-responsive lipid core micelles for tumour targeting.

A new acid-sensitive drug-delivery nanocarrier has been developed for tumour targeting. The self-assembling co-polymer stearoyl-PEG-poly-sulfadimethoxine methacrylate (stearoyl-PEG-polySDM) was prepared to obtain micelles with responsive behaviour in the physiopathologic pH range. Stearoyl-PEG-polySDM was synthesised using a multi-step procedure that includes pH-sensitive sulfadimethoxine methacrylate polymerisation by AGET-ATRP at the amino terminal side of stearoyl-PEG-NH2. Chemical analysis showed that the stearoyl-PEG-polySDM co-polymer contained a mean of seven methacryloyl sulfadimethoxines per molecule. Potentiometric and turbidimetric analyses showed that stearoyl-PEG-polySDM has an apparent pKa of 7.2 and a cloud point at pH 7.0. In water at pH 7.4, the co-polymer assembled spontaneously into 13.2±3.1 nm micelles with a critical micelle concentration (CMC) of 36 μM. Cell-culture studies showed that the material was more biocompatible with respect to the control Brij-700®. The paclitaxel loading capacity of the micelles was 3.25±0.25% (w/w, %). The colloidal formulations were stable at pH 7.4 for several hours, while at pH 6.5, they rapidly rearranged and aggregated. Fluorescence spectroscopic and cytofluorimetric studies showed that the incubation of MCF-7 tumour cells with fluorescein-labelled stearoyl-PEG-polySDM at pH 6.5 resulted in massive time-dependent cell association, while the incubation at pH 7.4 showed significantly lower cell interaction. Confocal microscopy confirmed that at pH 6.5, the micelles are taken up by cells and that the fluorescein-labelled stearoyl-PEG-polySDM is distributed into the cytosol. At pH 6.5, paclitaxel-loaded stearoyl-PEG-polySDM micelles had a higher cytotoxic effect than the micelles incubated at pH 7.4. The former displayed similar cytotoxic activity to free paclitaxel.

Synthesis and characterization of variable conformation pH responsive block co-polymers for nucleic acid delivery and targeted cell entry†

Responsive materials that change conformation with varying pH have been prepared from a range of amphiphilic block co-polymers. The individual blocks are composed of (a) permanently hydrophilic chains with neutral functionality and (b) acrylate polymers with weakly basic side-chains. Variation in co-monomer content, molar mass and block ratios/compositions leads to a range of pH-responses, manifest through reversible self-assembly into micelles and/or polymersomes. These transitions can be tuned to achieve environmental responses in a pH range from 5–7, as shown by turbidimetric analysis, NMR and dynamic light scattering measurements (DLS). Further characterization by transmission electron microscopy (TEM) indicates that polymersomes with diameters of 100–200 nm can be formed under certain pH-ranges where the weakly basic side-chains are deprotonated. The ability of the systems assembled with these polymers to act as pH-responsive containers is shown by DNA encapsulation and release studies, and their potential for application as vehicle for drug delivery is proved by cell metabolic activity and cell uptake measurements.

Star-Like Oligo-Arginyl-Maltotriosyl Derivatives as Novel Cell-Penetrating Enhancers for the Intracellular Delivery of Colloidal Therapeutic Systems

A novel nonpeptide, multiarmed oligo-arginyl derivative was engineered as a cell-penetration enhancer for the delivery of bioactive macromolecules and colloidal drug systems. Hepta-arginyl-maltotriosylamido-N-acetyl-dodecanoyl acid (Arg(7)-Malt-NAcC(12) acid) was synthesized through a carefully designed multistep chemical protocol, as follows: (1) maltotriose derivatization with 12-amino-dodecanoic acid and acetylation of the free amino group; (2) esterification of the maltotriosyl hydroxyl groups with 2-bromo-isobutyryl bromide; and (3) synthesis of star-like oligomer bearing multiple copies of arginine moieties under atom transfer radical polymerization (ATRP) conditions. The intermediates and final product were characterized by (1)H NMR, IR, mass spectrometry, colorimetric assays, and elemental analysis. Cytotoxicity studies on the final polymeric material showed that this novel cell-penetrating enhancer does not have significant toxic effects on MCF-7 and MC3T3-E1 cell lines. The IC(50) was greater than 100 μM with both cell lines, while the polyethylenimine with similar average molecular mass (M(n)) that was used as a reference showed an IC(50) of 30 and 40 μM, for MCF-7 and MC3T3-E1, respectively. The biological properties of the novel bioconjugate were investigated using a fluorescein-labeled bovine serum albumin (FITC-BSA) as a hydrophilic cargo model. MCF-7 and MC3T3-E1 cells were incubated for 60 min with the Arg(7)-Malt-NAcC(12)-conjugated FITC-BSA [(Arg(7)-Malt-NAcC(12))(2)-FITC-BSA] or FITC-BSA, and the intracellular fluorescence level was analyzed by spectrofluorimetric analysis of cell lysate, cytofluorimetry, and confocal microscopy. The fluorescence of the lysate of MCF-7 and MC3T3-E1 cells that were incubated with (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA at 37 °C was approximately 4.5 times higher than the fluorescence obtained with cells incubated with FITC-BSA. At 4 °C, the cell uptake of (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was only 2 times higher than that of FITC-BSA. Cytofluorimetric studies showed that, after (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA treatment, over 80% of MCF-7 cells and over 95% of MC3T3-E1 cells displayed enhanced fluorescence. Confocal investigations showed punctuated fluorescence within the cytosol in both cell lines, indicating that (Arg(7)-Malt-NAcC(12))(2)-FITC-BSA was confined to endosomes, with no fluorescence observed in the nucleus.

pH-responsive poly(4-hydroxybenzoyl methacrylates) – design and engineering of intelligent drug delivery nanovectors

In this study a novel class of pH-responsive polymers based on new substituted phenol monomers is presented. A judicious choice of the electron-withdrawing groups on the aromatic ring allowed modulation of both their pKa and hydrophobicity. Preliminary experiments using poly[2-((methacryloyloxy)ethyl-3-chloro-4-hydroxybenzoate)-r-(glycerol methacrylate)], poly(MCH-r-GMA), showed that the pKa of the MCH repeating units is in the 6.5–7 range, which opened the way for their application in the assembly of drug-delivery nanocarriers. These pH-responsive materials are unusual in that, unlike the systems based on organic amine frequently employed for this purpose, they possess hydrophilic behaviour in basic aqueous conditions, whilst they become hydrophobic upon acidification. Poly(MCH-b-PEGMA475) could be easily assembled in either stable micellar or polymersome nanocarriers by simply modifying the hydrophilic : hydrophobic balance of their block components. Interestingly, the size of poly(MCH-b-PEGMA475) nanoaggregates was found to be strongly pH-dependent, going from 32 nm at pH 7.4 to 120 nm at pH 5.5. Preliminary drug entrapment experiments showed a loading of tamoxifen model hydrophobic drug as high as 17–19% w/w, whilst as expected a significant amount of hydrophilic doxorubicin hydrochloride, 14% w/w, could be incorporated in polymersome poly(MCH-b-PEGMA475) nanocarriers.

Engineered Polymer-Transferrin Conjugates as Self-Assembling Targeted Drug Delivery Systems

Polymer-protein conjugates can be engineered to self-assemble into discrete and well-defined drug delivery systems, which combine the advantages of receptor targeting and controlled drug release. We designed specific conjugates of the iron-binding and transport protein, transferrin (Tf), to combine the advantages of this serum-stable protein as a targeting agent for cancer cells with self-assembling polymers to act as carriers of cytotoxic drugs. Tf variants were expressed with cysteine residues at sites spanning different regions of the protein surface, and the polymer conjugates grown from these variants were compared with polymer conjugates grown from nonselectively derivatized sites on native Tf. The resulting synthetic biopolymer hybrids were evaluated for self-assembly properties, size and topology, ability to carry an anticancer drug (paclitaxel), and cytotoxicity with and without a drug payload in a representative human colon cancer cell line. The results demonstrated that the engineered Tf variant polymer conjugates formed better-defined self-assembled nanoparticles than the nonselectively derivatized conjugates and showed greater efficacy in paclitaxel delivery. A polymer conjugate grown from a specific Tf variant, S415C was found to be taken up rapidly into cancer cells expressing the Tf-receptor, and, while tolerated well by cells in the absence of drugs, was as cytotoxic as free paclitaxel, when loaded with the drug. Importantly, the S415C conjugate polymer was not the most active variant in Tf-receptor binding, suggesting that the nanoscale self-assembly of the polymer-protein hybrid is also a key factor in delivery efficacy. The data overall suggest new design rules for polymer-biopolymer hybrids and therapeutic delivery systems, which include engineering specific residues for conjugation that mediate nanoscale assembly as well as control of ligand-receptor interactions to target specific cell types.

Re-programming pullulan for targeting and controlled release of doxorubicin to the hepatocellular carcinoma cells

ABSTRACT A novel bioconjugate for hepatocellular carcinoma (HCC) targeting was obtained by pullulan re‐programming, which involves the backbone oxidation and conjugation of targeting peptide and doxorubicin (Doxo) through a releasable linker. Preliminary in vivo studies showed that the oxidation of 40 glucopyranose units (GPU) out of 100 remarkably reduced the pullulan unspecific liver tropism. This oxidized polymer was functionalized with PreS1 to selectively target the HCC and with rhodamine (Rhod) as label to perform in vitro cell up‐take investigations. PreS1 and Rhod were conjugated to the aldehydes present along the oxidized pullulan backbone through a 3.4 and 2 kDa PEG spacer, respectively, and by reductive amination. The resulting PreS1‐Pull‐Rhod contained a mean of 8 PreS1 per oxidized pullulan chain. Cell culture studies were performed by using HepG2/SERPINB3 cells that overexpress the serpine B3 receptor and control HepG2/EMPTY cells that do not overexpress the receptor. A comparative study by cytofluorimetry and confocal microscopy performed using PreS1‐Pull‐Rhod and Pull‐Rhod (control polymer) showed that PreS1 conveys to the conjugate high cell selectivity. Afterwards, the oxidized pullulan was exploited to generate a targeted drug delivery system by conjugation of Doxo to the polymer backbone through a hydrazone pH‐sensitive bond and NH2‐PEG3.4 kDa‐PreS1. The PreS1‐Pull‐Doxo conjugate showed a two‐fold increase of anticancer activity with respect to the control Pull‐Doxo towards HepG2/SERPINB3 cells.

One-pot RAFT and fast polymersomes assembly: A 'beeline' from monomers to drug-loaded nanovectors

Rapid and simple routes to functional polymersomes are increasingly needed to expand their clinical or industrial applications. Here we describe a novel strategy where polymersomes are prepared through an in-line process in just a few hours, starting from simple acrylate or acrylamide monomers. Using Perriers protocol, well-defined amphiphilic diblock copolymers formed from PEG acrylate (mPEGA480), 2-(acryloyloxy)ethyl-3-chloro-4-hydroxybenzoate (ACH) or 2-(3-chloro-4-hydroxybenzamido)ethyl acrylate (CHB), have been synthesised by RAFT polymerisation in one-pot, pushing the monomer conversion for each block close to completion (≥94%). The reaction mixture, consisting of green biocompatible solvents (ethanol/water) have then been directly utilised to generate well-defined polymersomes, by simple cannulation into water or in a more automated process, by using a bespoke microfluidic device. Terbinafine and cyanocobalamine were used to demonstrate the suitability of the process to incorporate model hydrophobic and hydrophilic drugs, respectively. Vesicles size and morphology were characterised by DLS, TEM, and AFM. In this work we show that materials and experimental conditions can be chosen to allow facile and rapid generation drug-loaded polymersomes, through a suitable in-line process, directly from acrylate or acrylamide monomer building blocks.

New molecular targets for functionalized nanosized drug delivery systems in personalized therapy for hepatocellular carcinoma

&NA; Hepatocellular carcinoma, the most frequent solid tumor of the liver, has a very poor prognosis, being the second most common cause of death from cancer worldwide. The incidence and mortality of this liver tumor are increasing in most areas of the world as a consequence of aging and the emerging of new risk factors such as the metabolic syndrome, beside the recognized role of hepatitis B and C viral infections and alcohol abuse. Despite the increasing knowledge on the molecular mechanisms underlying hepatic carcinogenesis, effective therapeutic strategies are still an unmet clinical need. Efforts have been made to develop selective drugs as well as effective targeted drug delivery systems. The development of novel drug carriers for therapeutic molecules can indeed offer a valuable strategy to ameliorate the efficacy of HCC treatment. In this review, we discuss recent drug delivery strategies for HCC treatment based on the exploitation of targeted nanoparticles (NPs). Indeed, a few of these platforms have achieved an advanced stage of preclinical development. Here, we review the most promising drug nanovehicles based on both synthetic and natural polymers, including polysaccharides that have emerged for their biocompatibility and biodegradability. To maximize site‐selectivity and therapeutic efficacy, drug delivery systems should be functionalized with ligands which can specifically recognize and bind targets expressed by HCC, namely cell membrane associated antigens, receptors or biotransporters. Cell surface and intracellular molecular targets are exploited either to selectively deliver drug‐loaded nanovehicles or to design novel selective therapeutics. In conclusion, the combination of novel and safe drug delivery strategies based on site‐specific targeted drug nanovehicles with therapeutic molecular targets may significantly improve the pharmacological efficacy for the treatment of HCC. Graphical abstract Figure. No caption available.