In order to develop targeted pharmaceutical carriers additionally capable of responding certain local stimuli, such as decreased pH values in tumors or infarcts, targeted long-circulating PEGylated liposomes and PEG-phosphatidylethanolamine (PEG-PE)-based micelles have been prepared with several functions. First, they are capable of targeting a specific cell or organ by attaching the monoclonal antimyosin antibody 2G4 to their surface via pNP-PEG-PE moieties. Second, these liposomes and micelles were additionally modified with biotin or TAT peptide (TATp) moieties attached to the surface of the nanocarrier by using biotin-PE or TATp-PE or TATp-short PEG-PE derivatives. PEG-PE used for liposome surface modification or for micelle preparation was made degradable by inserting the pH-sensitive hydrazone bond between PEG and PE (PEG-Hz-PE). Under normal pH values, biotin and TATp functions on the surface of nanocarriers were "shielded" by long protecting PEG chains (pH-degradable PEG 2000 -PE or PEG 5000 -PE) or by even longer pNP-PEG-PE moieties used to attach antibodies to the nanocarrier (non-pH-degradable PEG 3400 -PE or PEG 5000 -PE). At pH 7.5-8.0, both liposomes and micelles demonstrated high specific binding with 2G4 antibody substrate, myosin, but very limited binding on an avidin column (biotin-containing nanocarriers) or internalization by NIH/3T3 or U-87 cells (TATp-containing nanocarriers). However, upon brief incubation (15-to-30 min) at lower pH values (pH 5.0-6.0) nanocarriers lost their protective PEG shell because of acidic hydrolysis of PEG-Hz-PE and acquired the ability to become strongly retained on avidin-column (biotin-containing nanocarriers) or effectively internalized by cells via TATp moieties (TATp-containing nanocarriers). We consider this result as the first step in the development of multifunctional stimuli-sensitive pharmaceutical nanocarriers.

Over the last few decades, nanocarriers for drug delivery have emerged as powerful tools with unquestionable potential to improve the therapeutic efficacy of anticancer drugs. Many colloidal drug delivery systems are underdevelopment to ameliorate the site specificity of drug action and reduce the systemic side effects. By virtue of their small size they can be injected intravenously and disposed into the target tissues where they release the drug. Nanocarriers interact massively with the surrounding environment, namely, endothelium vessels as well as cells and blood proteins. Consequently, they are rapidly removed from the circulation mostly by the mononuclear phagocyte system. In order to endow nanosystems with long circulation properties, new technologies aimed at the surface modification of their physicochemical features have been developed. In particular, stealth nanocarriers can be obtained by polymeric coating. In this paper, the basic concept underlining the “stealth” properties of drug nanocarriers, the parameters influencing the polymer coating performance in terms of opsonins/macrophages interaction with the colloid surface, the most commonly used materials for the coating process and the outcomes of this peculiar procedure are thoroughly discussed.

Despite the tremendous technical advancements in 3D bioprinting, the concept of fabricating 3D structures and functional tissues directly in live animals remains a visionary challenge. We show that 3D cell-laden hydrogels can be efficiently bioprinted across tissues and within tissues of living animals.We developed photo-sensitive polymers that allow in vitro and in vivo fabrication of hydrogels into pre-existing structures, by bio-orthogonal two-photon cycloaddition and crosslinking at wavelengths longer than 850 nm, without byproducts. By this technique, that we name intravital 3D bioprinting, after injection of these polymers in vivo it is possible to fabricate complex 3D structures inside tissues of living mice, including the dermis across epidermis, the skeletal muscle across epimysium or the brain across meninges. The use of commonly available multi-photon microscopes allows accurate (XYZ) positioning and orientation of bioprinted structures into specific anatomical sites. Finally, we show that intravital 3D bioprinting of donor muscle-derived stem cells allows de novo formation of myofibers in host animals. We envision that this strategy will offer an alternative in vivo approach to conventional bioprinting technology, holding great promises to substantially change the paradigm of 3D bioprinting for pre-clinical and clinical use.

Despite the substantial body of research investigating the use of liposomes, niosomes and other bilayer vesicles for drug delivery, the translation of these systems into licensed products remains limited. Indeed, recent shortages in the supply of liposomal products demonstrate the need for new scalable production methods for liposomes. Therefore, the aim of our research has been to consider the application of microfluidics in the manufacture of liposomes containing either or both a water soluble and a lipid soluble drug to promote co-delivery of drugs. For the first time, we demonstrate the entrapment of a hydrophilic and a lipophilic drug (metformin and glipizide respectively) both individually, and in combination, using a scalable microfluidics manufacturing system. In terms of the operating parameters, the choice of solvents, lipid concentration and aqueous:solvent ratio all impact on liposome size with vesicle diameter ranging from ∼90 to 300nm. In terms of drug loading, microfluidics production promoted high loading within ∼100nm vesicles for both the water soluble drug (20-25% of initial amount added) and the bilayer embedded drug (40-42% of initial amount added) with co-loading of the drugs making no impact on entrapment efficacy. However, co-loading of glipizide and metformin within the same liposome formulation did impact on the drug release profiles; in both instances the presence of both drugs in the one formulation promoted faster (up to 2 fold) release compared to liposomes containing a single drug alone. Overall, these results demonstrate the application of microfluidics to prepare liposomal systems incorporating either or both an aqueous soluble drug and a bilayer loaded drug.

Surface decoration of gold nanoparticles with thermoresponsive polymers endows a temperature tunable colloidal system switchable for enhanced intracellular up-take. Gold nanoparticles (AuNP, 18 ± 11 nm-diameter) produced by laser ablation synthesis in liquid solution were surface coated with thermoresponsive thiol terminated poly-N-isopropylacrylamide-co-acrylamide co-polymer possessing a lower critical solution temperature (LCST) at 37 °C. Under selected conditions about 3800 polymer chains were conjugated per particle. The polymer coated nanoparticles were found to display thermosensitive properties, as in solution they exhibited reversible aggregation/deaggregation above and below the LCST, respectively. Cell culture studies showed that the polymer decorated AuNP were located into human breast adenocarcinoma MCF7 cells treated at 40 °C (12000 AuNP/cell) with more than 80-fold greater up-take compared to cells treated at 34 °C with the same particles (140 AuN/cell). This difference is attributable to a ‘switching’ of the polymer coating to a globule state at 37 °C and an increased hydrophobicity of the particles with a simultaneous loss of the ‘stealth’ properties of the polymer coating. By contrast, cell up-take of uncoated AuNP (about 6000 AuNP/cell) did not depend on the incubation temperature. These data show that good control of the AuNP cell up-take can be obtained with the new polymer-gold nanoconjugates, and suggest that these systems might find use for targeting cells in vitro by a small temperature change or in vivo in body sites, such as inflamed or tumour tissues, where a temperature variation is already present

The application of atom transfer radical polymerization (ATRP) for preparation of a novel class of protein-polymer bioconjugates is described, exemplified by the synthesis of a recombinant human growth hormone (rh-GH) poly(ethylene glycol) methyl ether methacrylate (PEGMA) hybrid. The rh-GH protein was activated via a bromo-ester functionalized linker and used as a macroinitiator to polymerize the hydrophilic monomer PEGMA under solely aqueous conditions at 4 degrees C. ATRP conditions resulted in controlled polymer growth from rh-GH with low-polydispersity polyPEGMA chains. The rh-GH PEGMA product exhibited properties consistent with the presence of attached hydrophilic polymer chains, namely, high stability to denaturation and proteolysis. The polymerization conditions and conjugation proceeded with retention of the biological activity of the hormone. The rh-GH PEGMA was administered subcutaneously to rats and the activity compared to native rh-GH. The rh-GH PEGMA exhibited similar activity as the native rh-GH in vivo when a daily dose of 40 microg was administered. However, when a higher dose of 120 microg was administered with 3 days between injections the bioavailability of the rh-GH PEGMA was significantly better than that of the native. The results therefore demonstrate that ATRP can be successfully used as a general alternative approach to direct polymer conjugation, namely, PEGylation, to produce PEG-like protein conjugates. This technique can be exploited to design and synthesize protein-polymer derivatives with tailored therapeutic properties.

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beta-Cyclodextrin-poly(ethylene glycol)-folic acid conjugate (CD-PEG-FA) was synthesized according to a two-step procedure: (1). synthesis of CD-PEG-NH(2) by reaction of monotosyl-activated beta-cyclodextrin with excess of 700 Da diamino-PEG; (2). synthesis of CD-PEG-FA by reaction of CD-PEG-NH(2) with succinimidyl ester-activated folic acid. The CD-PEG-NH(2) intermediate was purified by precipitation in acetone, and the CD-PEG-FA by gel permeation and C-18 reversed-phase chromatography. Both CD-PEG-NH(2) and CD-PEG-FA were analyzed by mass spectrometry, (1)H NMR, and UV-vis spectroscopy. All analytical methods confirmed the theoretical composition of the conjugates: the CD-PEG-NH(2) intermediate was composed of CD and PEG in the molar ratio of 1:1, and the CD-PEG-FA was composed of beta-cyclodextrin, PEG, and folic acid in the molar ratio of 1:1:1. The CD-PEG-FA conjugate was highly soluble in buffer (>42 mM) as compared to the unmodified beta-cyclodextrin (16.3 mM). Phase solubility diagrams of beta-estradiol revealed that drug solubility increases from 11 microM in buffer to 600 microM in the presence of beta-cyclodextrins and 5900 microM with CD-PEG-FA. However, the affinity of beta-estradiol for beta-cyclodextrins decreased about 4 times with PEG and folic acid conjugation. Stability studies carried out using chlorambucil confirmed that the conjugate partially prevents drug degradation in buffer, although this effect was considerably lower than that obtained with beta-cyclodextrin. Computer modeling studies showed that the folic acid linked to the beta-cyclodextrins through a PEG spacer could partially interact with the cyclodextrin cavity. Finally, CD-PEG-FA displayed reduced hemolytic effect as compared to unmodified beta-cyclodextrin.