Pullulan Based Bioconjugates for Ocular Dexamethasone Delivery

Posterior segment eye diseases are mostly related to retinal pathologies that require pharmacological treatments by invasive intravitreal injections. Reduction of frequent intravitreal administrations may be accomplished with delivery systems that provide sustained drug release. Pullulan-dexamethasone conjugates were developed to achieve prolonged intravitreal drug release. Accordingly, dexamethasone was conjugated to similar to 67 kDa pullulan through hydrazone bond, which was previously found to be slowly cleavable in the vitreous. Dynamic light scattering and transmission electron microscopy showed that the pullulan-dexamethasone containing 1:20 drug/glucose unit molar ratio (10% w/w dexamethasone) self-assembled into nanoparticles of 461 +/- 30 nm and 402 +/- 66 nm, respectively. The particles were fairly stable over 6 weeks in physiological buffer at 4, 25 and 37 degrees C, while in homogenized vitreous at 37 degrees C, the colloidal assemblies underwent size increase over time. The drug was released slowly in the vitreous and rapidly at pH 5.0 mimicking lysosomal conditions: 50% of the drug was released in about 2 weeks in the vitreous, and in 2 days at pH 5.0. In vitro studies with retinal pigment epithelial cell line (ARPE-19) showed no toxicity of the conjugates in the cells. Flow cytometry and confocal microscopy showed cellular association of the nanoparticles and intracellular endosomal localization. Overall, pullulan conjugates showed interesting features that may enable their successful use in intravitreal drug delivery.Peer reviewe

Physical PEGylation to Prevent Insulin Fibrillation

Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG5kDa-cholane and mPEG5kDa-cholesterol, respectively. Microcalorimetric analyses showed that mPEG5kDa-cholane and mPEG5kDa-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 104 and 1.14 105 M-1, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of \u3b1-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG5kDa-cholesterol has superior stabilizing effect of the protein conformation than mPEG5kDa-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG5kDa-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG5kDa-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG5kDa-cholane and mPEG5kDa-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation

Mannosylated Polycations Target CD206+Antigen-Presenting Cells and Mediate T-Cell-Specific Activation in Cancer Vaccination

Immunotherapy is deemed one of the most powerful therapeutic approaches to treat cancer. However, limited response and tumor specificity are still major challenges to address. Herein, mannosylated polycations targeting mannose receptor are developed as vectors for plasmid DNA (pDNA)-based vaccines to improve selective delivery of genetic material to antigen presenting cells and enhance immune cell activation. Three diblock glycopolycations (M15A12, M29A25, and M58A45) and two triblock copolymers (M29A29B9 and M62A52B32) are generated by using mannose (M), agmatine (A), and butyl (B) derivatives to target CD206, complex nucleic acids, and favor the endosomal escape, respectively. All glycopolycations efficiently complex pDNA at N/P ratiosPeer reviewe

pH-Controlled Liposomes for Enhanced Cell Penetration in Tumor Environment

An innovative pH-switchable colloidal system that can be exploited for site-selective anticancer drug delivery has been generated by liposome decoration with a new novel synthetic non-peptidic oligo-arginine cell-penetration enhancer (CPE) and a quenching PEGylated counterpart that detaches from the vesicle surface under the acidic conditions of tumors. The CPE module (Arg(4)-DAG) is formed by four arginine units conjugated to a first-generation (G1) 2,2-bis(hydroxymethyl)propionic acid (bis-MPA)/2,2-bis(aminomethyl)propionic acid (bis-AMPA) polyester dendron terminating with 1,2-distearoyl-3-azidopropane for liposome bilayer insertion. The zeta potential of the Arg(4)-DAG-decorated liposomes increased up to +32 mV as the Arg(4)-DAG/lipids molar ratio increased. The Arg(4)-DAG liposome shielding at pH 7.4 was provided by methoxy-PEGS(5 kDa)-polymethacryloyl sulfadimethoxine (mPEG(5) (kDa)-SDM8) with 7.1 apparent pK(a). Zeta potential, surface plasmon resonance and synchrotron small-angle X-ray scattering analyses showed that at pH 7.4 mPEG(5) (kDa)-SDM8 associates with polycationic Arg(4)-DAG-decorated liposomes yielding liposomes with neutral zeta potential. At pH 6.5, which mimics the tumor environment, mPEG(5) (kDa)-SDM8 detaches from the liposome surface yielding Arg(4)-DAG exposure. Flow cytometry and confocal microscopy showed a 30-fold higher HeLa cancer cell association of the Arg(4)-DAG-decorated liposomes compared to non-decorated liposomes. At pH 7.4, the mPEG(5) (kDa)-SDM8-coated liposomes undergo low cell association while remarkable cell association occurred at pH 6.5, which allowed for the controlled intracellular delivery of model macromolecules and small molecules loaded in the liposome under tumor conditions.Peer reviewe

Enhanced uptake in 2D- and 3D- lung cancer cell models of redox responsive PEGylated nanoparticles with sensitivity to reducing extra-and intracellular environments

In the treatment of lung cancer, there is an urgent need of innovative medicines to optimize pharmacological responses of conventional chemotherapeutics while attenuating side effects. Here, we have exploited some relatively unexplored subtle differences in reduction potential, associated with cancer cell microenvironments in addition to the well-known changes in intracellular redox environment. We report the synthesis and application of novel redox-responsive PLGA (poly(lactic-co-glycolic acid)) -PEG(polyethylene glycol) nanoparticles (RR-NPs) programmed to change surface properties when entering tumor microenvironments, thus enhance cell internalization of the particles and their drug cargo. The new co-polymers, in which PEG and PLGA were linked by ‘anchiomeric effector’ dithiylethanoate esters were synthesized by a combination of ring-opening polymerization and Michael addition reactions and employed to prepare NPs. Non redox-responsive nanoparticles (nRR-NPs) based on related PLGA-PEG copolymers were also prepared as comparators. Spherical NPs of around 120 nm diameter with a low polydispersity index and negative zeta potentials as well as good drug loading of docetaxel were obtained. The NPs showed prolonged stability in relevant simulated biological fluids and a high ability to penetrate an artificial mucus layer due to the presence of the external PEG coating. Stability, FRET and drug release studies in conditions simulating intracellular reductive environments demonstrated a fast disassembly of the external shell of the NPs, thus triggering on-demand drug release. FACS measurements and confocal microscopy showed increased and faster uptake of RR-NPs in both 2D- and 3D- cell culture models of lung cancer compared to nRR-NPs. In particular, the ‘designed-in’ reductive instability of RR-NPs in conditioned cell media, the fast PEG release in the extracellular compartment, as well as a diminution of uptake rate in control experiments where extracellular thiols were neutralized, suggested a partial extracellular release of the PEG fringe that promoted rapid internalization of the residual NPs into cells. Taken together, these results provide further evidence of the effectiveness of PEGylated reducible nanocarriers to permeate mucus layer barriers, and establish a new means to enhance cancer cell uptake of drug carriers by extra-and intra-cellular cleavage of protein-and cell-shielding hydrophilic blocks

Direct routes to functional RAFT agents from substituted N-alkyl maleimides

N-substituted maleimides have become an indispensable tool for the synthesis of bioconjugates and functional materials. Herein, we present three strategies for the incorporation of N-alkyl substituted maleimides into RAFT agents and show that these maleimide-derived CTAs can be used to easily introduce a range of chemical functionality at the β-position of polymer chains, resulting in α,β,ω-functional RAFT polymers. With both functional maleimides and RAFT agents that are increasingly available on the market, the approach presented in this study could facilitate the synthesis of end-functional macromolecules and will complement well the range of existing synthetic routes, including those utilising N-substituted maleimides, to functional polymeric materials

Control of targeting ligand display by pH-responsive polymers on gold nanoparticles mediates selective entry into cancer cells

Selective targeting of cells for intracellular delivery of therapeutics represents a major challenge for pharmaceutical intervention in disease. Here we show pH triggered receptor-mediated endocytosis of nanoparticles via surface ligand exposure. Gold nanoparticles were decorated with two polymers: a 2 kDa PEG with a terminal folate targeting ligand, and a di-block copolymer including a pH-responsive and a hydrophilic block. At the normal serum pH of 7.4, the pH-responsive block (apparent pKa of 7.1) displayed a hydrophilic extended conformation, shielding the PEG-folate ligands, which inhibited cellular uptake of the nanoparticles. Under pH conditions resembling those of the extracellular matrix around solid tumours (pH 6.5), protonation of the pH-responsive polymer triggered a coil-to-globule polymer chain contraction, exposing folate residues on the PEG chains. In line with this, endocytosis of folate-decorated polymer-coated gold nanoparticles in cancer cells overexpressing folate receptor was significantly increased at pH 6.5, compared with pH 7.4. Thus, the tumour acidic environment and high folate receptor expression was effectively exploited to activate cell binding and endocytosis of these nanoparticles. These data provide proof-of-concept for strategies enabling extracellular pH stimuli to selectively enhance cellular uptake of drug delivery vectors and their associated therapeutic cargo

Control of aggregation temperatures in mixed and blended cytocompatible thermoresponsive block co-polymer nanoparticles

A small library of thermoresponsive amphiphilic copolymers based on polylactide-block-poly((2-(2-methoxyethoxy)ethyl methacrylate)-co-(oligoethylene glycol methacrylate)) (PLA-b-P(DEGMA)-co-(OEGMA)), was synthesised by copper-mediated controlled radical polymerisation (CRP) with increasing ratios of OEGMA:DEGMA. These polymers were combined in two ways to form nanoparticles with controllable thermal transition temperatures as measured by particle aggregation. The first technique involved the blending of two (PLA-b-P(DEGMA)-co-(OEGMA)) polymers together prior to assembling NPs. The second method involved mixing pre-formed nanoparticles of single (PLA-b-P(DEGMA)-co-(OEGMA)) polymers. The observed critical aggregation temperature Tt did not change in a linear relationship with the ratios of each copolymer either in the nanoparticles blended from different copolymers or in the mitures of pre-formed nanoparticles. However, where co-polymer mixtures were based on (OEG)9MA ratios within 5-10 mole% , a linear relationship between (OEG)9MA composition in the blends and Tt was obtained. The data suggest that OEGMA-based copolymers are tunable over a wide temperature range given suitable co-monomer content in the linear polymers or nanoparticles. Moreover, the thermal transitions of the nanoparticles were reversible and repeatable, with the cloud point curves being essentially invariant across at least three heating and cooling cycles, and a selected nanoparticle formulation was found to be readily endocytosed in representative cancer cells and fibroblasts

Influence of folate-targeted gold nanoparticles on subcellular localization and distribution into lysosomes

The cell interaction, mechanism of cell entry and intracellular fate of surface decorated nanoparticles are known to be affected by the surface density of targeting agents. However, the correlation between nanoparticles multivalency and kinetics of the cell uptake process and disposition of intracellular compartments is complicated and dependent on a number of physicochemical and biological parameters, including the ligand, nanoparticle composition and colloidal properties, features of targeted cells, etc. Here, we have carried out an in-depth investigation on the impact of increasing folic acid density on the kinetic uptake process and endocytic route of folate (FA)-targeted fluorescently labelled gold nanoparticles (AuNPs). A set of AuNPs (15 nm mean size) produced by the Turkevich method was decorated with 0–100 FA-PEG3.5kDa-SH molecules/particle, and the surface was saturated with about 500 rhodamine-PEG2kDa-SH fluorescent probes. In vitro studies carried out using folate receptor overexpressing KB cells (KBFR-high) showed that the cell internalization progressively increased with the ligand surface density, reaching a plateau at 50:1 FA-PEG3.5kDa-SH/particle ratio. Pulse-chase experiments showed that higher FA density (50 FA-PEG3.5kDa-SH molecules/particle) induces more efficient particle internalization and trafficking to lysosomes, reaching the maximum concentration in lysosomes at 2 h, than the lower FA density of 10 FA-PEG3.5kDa-SH molecules/particle. Pharmacological inhibition of endocytic pathways and TEM analysis showed that particles with high folate density are internalized predominantly by a clathrin-independent process