Synthetic macromolecular peptide-mimetics with amino acid substructure residues as protein stabilising excipients.

The clinical use of protein and peptide biotherapeutics requires fabrication of stable products. This particularly concerns stability towards aggregation of proteins or peptides. Here, we tested a hypothesis that interactions between a synthetic peptide, which is an aggregation-prone region analogue, and its homologous sequence on a protein of interest, could be exploited to design excipients which stabilise the protein against aggregation. A peptide containing the analogue of lysozyme aggregation-prone region (GILQINSRW) was conjugated to a RAFT agent and used to initiate the polymerisation of N-hydroxyethyl acrylamide, generating a GILQINSRW-HEA90 polymer, which profoundly reduced lysozyme aggregation. Substitution of tryptophan in GILQINSRW with glycine, to form GILQINSRG, revealed that tryptophan is a critical amino acid in the protein stabilisation by GILQINSRW-HEA90. Accordingly, polymeric peptide-mimetics of tryptophan, phenylalanine and isoleucine, which are often present in aggregation-prone regions, were synthesized. These were based on synthetic oligomers of acrylamide derivatives of indole-3 acetic acid (IND), phenylacetic acid (PHEN), or 2-methyl butyric acid (MBA), respectively, conjugated with hydrophilic poly(N-hydroxyethyl acrylamide) blocks to form amphiphilic copolymers denoted as INDm-, PHENm- and MTBm-b-HEAn. These materials were tested as protein stabilisers and it was shown that solution properties and the abilities of these materials to stabilise insulin and the peptide IDR 1018 towards aggregation are dependent on the chemical nature of their side groups. These data suggest a structure-activity relationship, whereby the indole-based INDm-b-HEAn peptide-mimetic displays properties of a potential stabilising excipient for protein formulations.

Control of cell penetration enhancer shielding and endosomal escape-kinetics crucial for efficient and biocompatible siRNA delivery.

Although cationic liposomes are efficient carriers for nucleic acid delivery, their toxicity often hampers the clinical translation. Polyethylene glycol (PEG) coating has been largely used to improve their stability and reduce toxicity. Nevertheless, it has been found to decrease the transfection process. In order to exploit the advantages of cationic liposomes and PEG decoration for nucleic acid delivery, liposomes decorated with tetraArg-[G-1]-distearoyl glycerol (Arg4-DAG) dendronic oligo-cationic lipid enhancer (OCE) and PEG-lipid have been investigated. Non decorated or OCE-decorated lipoplexes (OCEfree-LPX and OCE-LPX, respectively) were obtained by lipid film hydration using oligonucleotide (ON) solutions. PEG and OCE/PEG decorated lipoplexes (PEG-OCEfree-LPX and PEG-OCE-LPX, respectively) were obtained by post-insertion of 2 or 5 kDa PEG-DSPE on preformed lipoplexes. The OCE decoration yielded lipoplexes with size of about 240 nm, 84% loading efficiency at 10 N/P ratio, ten times higher than OCEfree-LPX, and prevented the ON release when incubated with physiological heparin concentration or with plasma. The PEG decoration reduced the zeta potential, enhanced the lipoplex stability in serum and decreased both hemolysis and cytotoxicity, while it did not affect the lipoplex size and ON loading. With respect to OCEfree-LPX, the OCE-LPX remarkably associated with cells and were taken up by different cancer cell lines (HeLa and MDA-MB-231). Interestingly, 2 or 5 kDa PEG decoration did not reduce either the cell interaction or the cell up-take of the cationic lipoplexes. With siRNA as a payload, OCE enabled efficient internalization, but endosomal release was hampered. Post-transfection treatment with the lysosomotropic drug chloroquine allowed to identify the optimal time point for endosomal escape. Chloroquine treatment after 12 to 20 h of LPX pre-incubation enabled siRNA mediated target knockdown indicating that this is the time window of endo-lysosomal processing. This indicates that OCE can protect siRNA from lysosomal degradation for up to 20 h, as shown by these rescue experiments.

Sulfation at Glycopolymer Side Chains Switches Activity at the Macrophage Mannose Receptor (CD206) In Vitro and In Vivo.

The mannose receptor (CD206) is an endocytic receptor expressed by selected innate immune cells and nonvascular endothelium, which plays a critical role in both homeostasis and pathogen recognition. Although its involvement in the development of several diseases and viral infections is well established, molecular tools able to both provide insight on the chemistry of CD206-ligand interactions and, importantly, effectively modulate its activity are currently lacking. Using novel SO4-3-Gal-glycopolymers targeting its cysteine-rich lectin ectodomain, this study uncovers and elucidates a previously unknown mechanism of CD206 blockade involving the formation of stable intracellular SO4-3-Gal-glycopolymer-CD206 complexes that prevents receptor recycling to the cell membrane. Further, we show that SO4-3-Gal glycopolymers inhibit CD206 both in vitro and in vivo, revealing hitherto unknown receptor function and demonstrating their potential as CD206 modulators within future immunotherapies.

A mechanoresponsive nano-sized carrier achieves intracellular release of drug on external ultrasound stimulus.

Control over intracellular release of therapeutic compounds incorporated into nano-carriers will open new possibilities for targeted treatments of various diseases including cancer, and viral and bacterial infections. Here we report our study on mechanoresponsive nano-sized liposomes which, following internalization by cells, achieve intracellular delivery of encapsulated cargo on application of external ultrasound stimulus. This is demonstrated in a bespoke cell reporter system designed to assess free drug in cytoplasm. Biophysical analyses show that drug release is attributable to the action of a mechanoresponsive spiropyran-based compound embedded in the liposomal lipid membrane. Exposure to external ultrasound stimulus results in opening of the molecular structure of the embedded spiropyran, a consequent increase in liposomal lipid membrane fluidity, and size-dependent release of encapsulated model drugs, all pointing to lipid bilayer perturbation. The study hence illustrates feasibility of the proposed concept where intracellular drug release from mechanoresponsive liposomes can be triggered on demand by external ultrasound stimulus.

Tobramycin-loaded complexes to prevent and disrupt Pseudomonas aeruginosa biofilms.

Carbohydrate-based materials are increasingly investigated for a range of applications spanning from healthcare to advanced functional materials. Synthetic glycopolymers are particularly attractive as they possess low toxicity and immunogenicity and can be used as multivalent ligands to target sugar-binding proteins (lectins). Here, we utilised RAFT polymerisation to synthesize two families of novel diblock copolymers consisting of a glycopolymers block containing either mannopyranose or galactopyranose pendant units, which was elongated with sodium 2-acrylamido-2-methyl-1-propanesulfonate (AMPS) to generate a polyanionic block. The latter enabled complexation of cationic aminoglycoside antibiotic tobramycin through electrostatic interactions (loading efficiency in the 0.5-6.3 wt% range, depending on the copolymer). The resulting drug vectors were characterized by dynamic light scattering, zeta-potential, and transmission electron microscopy. Tobramycin-loaded complexes were tested for their ability to prevent clustering or disrupt biofilm of the Pseudomonas aeruginosa Gram-negative bacterium responsible for a large proportion of nosocomial infection, especially in immunocompromised patients. P. aeruginosa possesses two specific tetrameric carbohydrate-binding adhesins, LecA (PA-IL, galactose/N-acetyl-D-galactosamine-binding) and LecB (PA-IIL, fucose/mannose-binding), and the cell-associated and extracellular adhesin CdrA (Psl/mannose-binding) thus ideally suited for targeted drug delivery using sugar-decorated tobramycin-loaded complexes here developed. Both aliphatic and aromatic linkers were utilised to link the sugar pendant units to the polyacrylamide polymer backbone to assess the effect of the nature of such linkers on bactericidal/bacteriostatic properties of the complexes. Results showed that tobramycin-loaded complexes efficiently suppressed (40 to 60% of inhibition) in vitro biofilm formation in PAO1-L P. aeruginosa and that preferential targeting of PAO1-L biofilm can be achieved using mannosylated glycopolymer-b-AMPSm.

PEG-polyaminoacid based micelles for controlled release of doxorubicin: Rational design, safety and efficacy study.

A library of amphiphilic monomethoxypolyethylene glycol (mPEG) terminating polyaminoacid co-polymers able to self-assemble into colloidal systems was screened for the delivery and controlled release of doxorubicin (Doxo). mPEG-Glu/Leu random co-polymers were generated by Ring Opening Polymerization from 5 kDa mPEG-NH2 macroinitiator using 16:0:1, 8:8:1, 6:10:1, 4:12:1 γ-benzyl glutamic acid carboxy anhydride monomer/leucine N-carboxy anhydride monomer/PEG molar ratios. Glutamic acid was selected for chemical conjugation of Doxo, while leucine units were introduced in the composition of the polyaminoacid block as spacer between adjacent glutamic repeating units to minimize the steric hindrance that could impede the Doxo conjugation and to promote the polymer self-assembly by virtue of the aminoacid hydrophobicity. The benzyl ester protecting the γ-carboxyl group of glutamic acid was quantitatively displaced with hydrazine to yield mPEG5kDa-b-(hydGlum-r-Leun). Doxo was conjugated to the diblock co-polymers through pH-sensitive hydrazone bond. The Doxo derivatized co-polymers obtained with a 16:0:1, 8:8:1, 6:10:1 Glu/Leu/PEG ratios self-assembled into 30-40 nm spherical nanoparticles with neutral zeta-potential and CMC in the range of 4-7 µM. At pH 5.5, mimicking endosome environment, the carriers containing leucine showed a faster Doxo release than at pH 7.4, mimicking the blood conditions. Doxo-loaded colloidal formulations showed a dose dependent cytotoxicity on two cancer cell lines, CT26 murine colorectal carcinoma and 4T1 murine mammary carcinoma with IC50 slightly higher than those of free Doxo. The carrier assembled with the polymer containing 6:10:1 hydGlu/Leu/PEG molar ratio {mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10]} was selected for subsequent in vitro and in vivo investigations. Confocal imaging on CT26 cell line showed that intracellular fate of the carrier involves a lysosomal trafficking pathway. The intratumor or intravenous injection to CT26 and 4T1 subcutaneous tumor bearing mice yielded higher antitumor activity compared to free Doxo. Furthermore, mPEG5kDa-b-[(Doxo-hydGlu)6-r-Leu10] displayed a better safety profile when compared to commercially available Caelyx®.

Combining Inducible Lectin Expression and Magnetic Glyconanoparticles for the Selective Isolation of Bacteria from Mixed Populations.

The selective isolation of bacteria from mixed populations has been investigated in varied applications ranging from differential pathogen identification in medical diagnostics and food safety to the monitoring of microbial stress dynamics in industrial bioreactors. Selective isolation techniques are generally limited to the confinement of small populations in defined locations, may be unable to target specific bacteria, or rely on immunomagnetic separation, which is not universally applicable. In this proof-of-concept work, we describe a novel strategy combining inducible bacterial lectin expression with magnetic glyconanoparticles (MGNPs) as a platform technology to enable selective bacterial isolation from cocultures. An inducible mutant of the type 1 fimbriae, displaying the mannose-specific lectin FimH, was constructed in Escherichia coli allowing for "on-demand" glycan-binding protein presentation following external chemical stimulation. Binding to glycopolymers was only observed upon fimbrial induction and was specific for mannosylated materials. A library of MGNPs was produced via the grafting of well-defined catechol-terminal glycopolymers prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization to magnetic nanoparticles. Thermal analysis revealed high functionalization (≥85% polymer by weight). Delivery of MGNPs to cocultures of fluorescently labeled bacteria followed by magnetic extraction resulted in efficient depletion of type 1 fimbriated target cells from wild-type or afimbriate E. coli. Extraction efficiency was found to be dependent on the molecular weight of the glycopolymers utilized to engineer the nanoparticles, with MGNPs decorated with shorter Dopa-(ManAA)50 mannosylated glycopolymers found to perform better than those assembled from a longer Dopa-(ManAA)200 analogue. The extraction efficiency of fimbriated E. coli was also improved when the counterpart strain did not harbor the genetic apparatus for the expression of the type 1 fimbriae. Overall, this work suggests that the modulation of the genetic apparatus encoding bacterial surface-associated lectins coupled with capture through MGNPs could be a versatile tool for the extraction of bacteria from mixed populations.

Thermosensitive "Smart" Surfaces for Biorecognition Based Cell Adhesion and Controlled Detachment.

The biorecognition-based control of attachment/detachment of MCF-7 cancer cells from polymer-coated surfaces is demonstrated. A glass surface is coated with a thermoresponsive statistical copolymer of poly(N-isopropylacrylamide-co-acrylamide) [p(NIPAm-co-Am)], which is end-capped with the Gly-Arg-Gly-Asp-Ser (GRGDS) peptide, and the hydrophilic polymer poly(ethylene glycol) (PEG). Below the lower critical solution temperature (LCST) of p(NIPAm-co-Am) (38 °C), the copolymers are in the extended conformation, allowing for accessibility of the GRGDS peptides to membrane-associated integrins thus enabling cell attachment. Above the LCST, the p(NIPAm-co-Am) polymers collapse into globular conformations, resulting in the shielding of the GRGDS peptides into the PEG brush with consequent inaccessibility to cell-surface integrins, causing cell detachment. The surface coating is carried out by a multi-step procedure that included: glass surface amination with 3-aminopropyltriethoxysilane; reaction of mPEG5kDa -N-hydroxysuccinimide (NHS) and p(NIPam-co-Am)15.1kDa -bis-NHS with the surface aminopropyl groups and conjugation of GRGDS to the carboxylic acid termini of p(NIPam-co-Am)15.1kDa -COOH. A range of spectrophotometric, surface, and microscopy assays confirmed the identity of the polymer-coated substrates. Competition studies prove that MCF-7 cancer cells are attached via peptide recognition at the coated surfaces according to the mPEG5kDa /p(NIPam-co-Am)15.1kDa -GRGDS molar ratio. These data suggest the system can be exploited to modulate cell integrin/GRGDS binding for controlled cell capture and release.

Polyvalent Diazonium Polymers Provide Efficient Protection of Oncolytic Adenovirus Enadenotucirev from Neutralizing Antibodies while Maintaining Biological Activity In Vitro and In Vivo.

Oncolytic viruses offer many advantages for cancer therapy when administered directly to confined solid tumors. However, the systemic delivery of these viruses is problematic because of the host immune response, undesired interactions with blood components, and inherent targeting to the liver. Efficacy of systemically administered viruses has been improved by masking viral surface proteins with polymeric materials resulting in modulation of viral pharmacokinetic profile and accumulation in tumors in vivo. Here we describe a new class of polyvalent reactive polymer based on poly( N-(2-hydroxypropyl)methacrylamide) (polyHPMA) with diazonium reactive groups and their application in the modification of the chimeric group B oncolytic virus enadenotucirev (EnAd). A series of six copolymers with different chain lengths and density of reactive groups was synthesized and used to coat EnAd. Polymer coating was found to be extremely efficient with concentrations as low as 1 mg/mL resulting in complete (>99%) ablation of neutralizing antibody binding. Coating efficiency was found to be dependent on both chain length and reactive group density. Coated viruses were found to have reduced transfection activity both in vitro and in vivo, with greater protection against neutralizing antibodies resulting in lower transgene production. However, in the presence of neutralizing antibodies, some in vivo transgene expression was maintained for coated virus compared to the uncoated control. The decrease in transgene expression was found not to be solely due to lower cellular uptake but due to reduced unpackaging of the virus within the cells and reduced replication, indicating that the polymer coating does not cause permanent inactivation of the virus. These data suggest that virus activity may be modulated by the appropriate design of coating polymers while retaining protection against neutralizing antibodies.

Water-soluble substituted chitosan derivatives as technology platform for inhalation delivery of siRNA.

Despite research efforts full potential of siRNA-based therapeutics has not yet been fully realized due to a need for suitable, effective delivery formulations. Here, we examine a potential of a new class of water-soluble chitosans as siRNA platform for pulmonary delivery. The system is based on piperazine-substituted chitosans, a material designed to integrate established, safe application of chitosan for mucosal administration with novel properties: the piperazine-substituted chitosans are freely water-soluble at physiological pH, possess low cytotoxicity (no significant reduction in cell viability up to 0.1 mg/ml), and provide efficient incorporation of siRNA into sub-300 nm colloidal complexes (at relatively low polymer/siRNA ratio of 5:1). In vitro, the complexes achieved silencing of a model gene at a level of 40-80%, when tested in a panel of lung epithelial cells. Considering the formulation 'developability', there were no significant changes in the complexes' size and integrity on aerosolisation by microsprayer (PenCentury™) device. Following intratracheal aerolisation, the complexes deposited throughout the lung, although relatively inhomogeneously, as judged from IVIS imaging of the isolated mouse lung (visualizing DY647-siRNA). In vivo data illustrate absence of adverse effects on repeated administration of complexes and significant tumor reduction in atopical lung cancer model in mice. Altogether, the data illustrates potential of substituted chitosan derivatives to be utilized as a safe system for inhalation delivery of siRNA.

Switching of Macromolecular Ligand Display by Thermoresponsive Polymers Mediates Endocytosis of Multiconjugate Nanoparticles.

Ligand-mediated targeting and internalization of plasma membrane receptors is central to cellular function. These types of receptors have accordingly been investigated as targets to facilitate entry of diagnostic and therapeutic constructs into cells. However, there remains a need to characterize how receptor targeting agents on nanoparticles interact at surface receptors and whether it is possible to control these interactions via exogenous stimuli. Here, we describe the switchable display of the iron-transporting protein, transferrin (Tf), at the surface of thermoresponsive polymer-coated gold nanoparticles and show that internalization of the coated nanoparticles into target cells changes across temperature ranges over which transferrin is expected to be sterically "hidden" by an extended polymer chain and then "revealed" by polymer chain collapse. The switching process is dependent on the numbers of transferrin molecules and thermoresponsive polymer chains attached and whether the assay temperature is above or below the transition temperatures of the responsive polymers at the nanoparticle surfaces. Significantly, however, the control of internalization is critically reliant on overall nanoparticle colloidal stability while the thermoresponsive component of the surface undergoes conformational change. The data show that the cell entry function of complex and large biomolecule ligands can be modulated by polymer-induced accessibility change but that a simple "hide and reveal" mechanism for ligand display following polymer chain collapse is insufficient to account for nanoparticle uptake and subsequent intracellular trafficking.

Dry-powder formulations of non-covalent protein complexes with linear or miktoarm copolymers for pulmonary delivery.

Pulmonary delivery of protein therapeutics has considerable clinical potential for treating both local and systemic diseases. However, poor protein conformational stability, immunogenicity and protein degradation by proteolytic enzymes in the lung are major challenges to overcome for the development of effective therapeutics. To address these, a family of structurally related copolymers comprising polyethylene glycol, mPEG2k, and poly(glutamic acid) with linear A-B (mPEG2k-lin-GA) and miktoarm A-B3 (mPEG2k-mik-(GA)3) macromolecular architectures was investigated as potential protein stabilisers. These copolymers form non-covalent nanocomplexes with a model protein (lysozyme) which can be formulated into dry powders by spray-drying using common aerosol excipients (mannitol, trehalose and leucine). Powder formulations with excellent aerodynamic properties (fine particle fraction of up to 68%) were obtained with particle size (D50) in the 2.5 µm range, low moisture content (<5%), and high glass transitions temperatures, i.e. formulation attributes all suitable for inhalation application. In aqueous medium, dry powders rapidly disintegrated into the original polymer-protein nanocomplexes which provided protection towards proteolytic degradation. Taken together, the present study shows that dry powders based on (mPEG2k-polyGA)-protein nanocomplexes possess potentials as an inhalation delivery system.

Polymers for binding of the gram-positive oral pathogen Streptococcus mutans.

Streptococcus mutans is the most significant pathogenic bacterium implicated in the formation of dental caries and, both directly and indirectly, has been associated with severe conditions such as multiple sclerosis, cerebrovascular and peripheral artery disease. Polymers able to selectively bind S. mutans and/or inhibit its adhesion to oral tissue in a non-lethal manner would offer possibilities for addressing pathogenicity without selecting for populations resistant against bactericidal agents. In the present work two libraries of 2-(dimethylamino)ethyl methacrylate (pDMAEMA)-based polymers were synthesized with various proportions of either N,N,N-trimethylethanaminium cationic- or sulfobetaine zwitterionic groups. These copolymers where initially tested as potential macromolecular ligands for S. mutans NCTC 10449, whilst Escherichia coli MG1655 was used as Gram-negative control bacteria. pDMAEMA-derived materials with high proportions of zwitterionic repeating units were found to be selective for S. mutans, in both isolated and S. mutans-E. coli mixed bacterial cultures. Fully sulfobetainized pDMAEMA was subsequently found to bind/cluster preferentially Gram-positive S. mutans and S. aureus compared to Gram negative E. coli and V. harveyi. A key initial stage of S. mutans pathogenesis involves a lectin-mediated adhesion to the tooth surface, thus the range of potential macromolecular ligands was further expanded by investigating two glycopolymers bearing α-mannopyranoside and ß-galactopyranoside pendant units. Results with these polymers indicated that preferential binding to either S. mutans or E. coli can be obtained by modulating the glycosylation pattern of the chosen multivalent ligands without incurring unacceptable cytotoxicity in a model gastrointestinal cell line. Overall, our results allowed to identify a structure-property relationship for the potential antimicrobial polymers investigated, and suggest that preferential binding to Gram-positive S. mutans could be achieved by fine-tuning of the recognition elements in the polymer ligands.

Synthesis, characterization and evaluation of in vitro toxicity in hepatocytes of linear polyesters with varied aromatic and aliphatic co-monomers.

Polyesters are extensively used in drug delivery because of their controllable biodegradation properties and perceived favorable cytocompatibility. However, new ester-based materials are continually being sought which can be produced from readily accessible monomers, which can be tuned for drug encapsulation and which retain good cellular compatibilities. In this study, 5 polyesters of similar molar mass were synthesized by reacting 1,10-decanediol with different ratios of succinic acid/phenylsuccinic acid and the effect of the phenyl side-chain group addition on polymer properties relevant to drug delivery was investigated. A polymer with a 70/30 ratio of succinic acid and phenylsuccinic acid was selected based on its ability to encapsulate a model dye in nanoparticle (NP) formulations, and was found to be slowly degradable in phosphate buffered saline (PBS) but more rapidly degraded in the presence of a lipase. The compatibility of NP formulations of this polymer either with or without a Pluronic F68 stabilizing coating was assessed in vitro using the C3A hepatocyte cell line. Cell viability was assessed, at NP concentrations ranging from 4.68-300µgmL-1 24h post-exposure, using the Alamar Blue, CDFA and Neutral Red assays. C3A cells internalized both coated and uncoated polyester NPs to a similar extent, with uptake observed to increase over time (10-1440min). Although cell viability was >80% at the concentrations tested, in all assays, it was found that a Pluronic F68 coated poly (decanediol-phenylsuccinate-co-succinate) stimulated significant DNA damage driven by an oxidant mechanism, whereas the non-coated polyester analogue and the Pluronic F68 alone had no effect. The results obtained suggest that new polyesters can be synthesized with desirable properties from the materials perspective but formulation with additional excipients requires careful evaluation for drug delivery applications.

Relation between detection rate and inappropriate shocks in single versus dual chamber cardioverter-defibrillator--an analysis from the OPTION trial.

The programming of implantable cardioverter-defibrillators (ICDs) influences inappropriate shock rates. The aim of the study is to analyse rates of patients with appropriate and inappropriate shocks according to detection zones in the OPTION trial. All patients received dual chamber (DC) ICDs randomly assigned to be programmed either to single chamber (SC) or to DC settings including PARAD+ algorithm. In a post-hoc analysis, rates of patients with inappropriate and appropriate shocks were calculated for shocks triggered at heart rates ≥ 170 bpm (ventricular tachycardia zone) and at rates ≥ 200 bpm (ventricular fibrillation zone). In the SC group, higher rates of patients with total and inappropriate shocks were delivered at heart rates ≥ 170 bpm than at rates ≥ 200 bpm (total shocks: 21.1% vs. 16.6%; p = 0.002; inappropriate shocks: 7.6% vs. 4.5%, p = 0.016; appropriate shocks: 15.2% vs. 13.5%; p = n.s.). No such differences were observed in the DC group (total shocks: 14.3% vs. 12.6%; p = n.s.; inappropriate shocks: 3.9% vs. 3.6%; p = n.s.; appropriate shocks: 12.2% vs. 10.4%; p = n.s.). The higher frequency of patients with total shocks with SC settings than with DC settings that benefit from PARAD+ was driven by a higher percentage of patients with inappropriate shocks in the VT zone (170-200 bpm) in the SC population.

Properties of acyl modified poly(glycerol-adipate) comb-like polymers and their self-assembly into nanoparticles.

There is an increasing need to develop bio-compatible polymers with an increased range of different physicochemical properties. Poly(glycerol-adipate) (PGA) is a biocompatible, biodegradable amphiphilic polyester routinely produced from divinyl adipate and unprotected glycerol by an enzymatic route, bearing a hydroxyl group that can be further functionalized. Polymers with an average Mn of ∼13 kDa can be synthesized without any post-polymerization deprotection reactions. Acylated polymers with fatty acid chain length of C4, C8, and C18 (PGAB, PGAO, and PGAS, respectively) at different degrees of substitution were prepared. These modifications yield comb-like polymers that modulate the amphiphilic characteristics of PGA. This novel class of biocompatible polymers has been characterized through various techniques such as FT-IR, 1H NMR, surface, thermal analysis, and their ability to self-assemble into colloidal structures was evaluated by using DLS. The highly tunable properties of PGA reported herein demonstrate a biodegradable polymer platform, ideal for engineering solid dispersions, nanoemulsions, or nanoparticles for healthcare applications. © 2016 The Authors. Journal of Polymer Science Part A: Polymer Chemistry Published by Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016, 54, 3267-3278.

Amphiphilic block copolymers from a renewable ε-decalactone monomer: prediction and characterization of micellar core effects on drug encapsulation and release.

Here we describe a methoxy poly(ethyleneglycol)-b-poly(ε-decalactone) (mPEG-b-PεDL) copolymer and investigate the potential of the copolymer as a vehicle for solubilisation and sustained release of indomethacin (IND). The indomethacin loading and release from mPEG-b-PεDL micelles (amorphous cores) was compared against methoxy poly(ethyleneglycol)-b-poly(ε-caprolactone)(mPEG-b-PCL) micelles (semicrystalline cores). The drug-polymer compatibility was determined through a theoretical approach to predict drug incorporation into hydrated micelles. Polymer micelles were prepared by solvent evaporation and characterised for size, morphology, indomethacin loading and release. All the formulations generated spherical micelles but significantly larger mPEG-b-PεDL micelles were observed compared to mPEG-b-PCL micelles. A higher compatibility of the drug was predicted for PCL cores based on Flory-Huggins interaction parameters (χsp) using the Hansen solubility parameter (HSP) approach, but higher measured drug loadings were found in micelles with PεDL cores compared to PCL cores. This we attribute to the higher amorphous content in the PεDL-rich regions which generated higher micellar core volumes. Drug release studies showed that the semicrystalline PCL core was able to release IND over a longer period (80% drug release in 110 h) compared to PεDL core micelles (80% drug release in 72 h).

Phosphonium Polymethacrylates for Short Interfering RNA Delivery: Effect of Polymer and RNA Structural Parameters on Polyplex Assembly and Gene Knockdown.

Synthetic polymers containing quaternary phosphonium salts are an emerging class of materials for the delivery of oligo/polynucleotides. In this work, cationic phosphonium salt-containing polymethacrylates and their corresponding ammonium analogues were synthesized by reversible addition-fragmentation chain transfer polymerization. Both the nature of the charged heteroatom (N vs P) and the length of the spacer separating the cationic units along the polymer backbone (oxyethylene vs trioxyethylene) were systematically varied. Polymers efficiently bound short interfering RNA (siRNA) at N(+)/P(-) or P(+)/P(-) ratios of 2 and above. At a 20:1 ratio, small polyplexes (Rh: 4-15 nm) suitable for cellular uptake were formed that displayed low cytotoxicity. While siRNA polyplexes from both ammonium and phosphonium polymers were efficiently internalized by green fluorescent protein (GFP)-expressing 3T3 cells, no knockdown of GFP expression was observed. However, 65% Survivin gene knockdown was observed when siRNA was replaced with novel, multimerized long interfering RNA in HeLa cells, demonstrating the importance of RNA macromolecular architecture on RNA-mediated gene silencing.

Triblock Copolymer Nanovesicles for pH-Responsive Targeted Delivery and Controlled Release of siRNA to Cancer Cells.

New pH-responsive polymersomes for active anticancer oligonucleotide delivery were prepared from triblock copolymers. The delivery systems were formed by two terminal hydrophilic blocks, PEG and polyglycerolmethacrylate (poly-GMA), and a central weakly basic block, polyimidazole-hexyl methacrylate (poly-ImHeMA), which can complex with oligonucleotides and control vesicle formation/disassembly via pH variations. Targeted polymersomes were prepared by mixing folate-derivatized and underivatized copolymers. At pH 5, ds-DNA was found to complex with the pH-responsive copolymers at a N/P molar ratio above ∼2:1, which assisted the encapsulation of ds-DNA in the polymersomes, while low association was observed at pH 7.4. Cytotoxicity studies performed on folate receptor overexpressing KB and B16-F10 cells and low folate receptor expressing MCF-7 cells showed high tolerance of the polymersomes at up to 3 mg/mL concentration. Studies performed with red blood cells showed that at pH 5.0 the polymersomes have endosomolytic properties. Cytofluorimetric studies showed a 5.5-fold higher uptake of ds-DNA loaded folate-functional polymersomes in KB cells compared to nontargeted polymersomes. In addition, ds-DNA was found to be localized both in the nucleus and in the cytosol. The incubation of luciferase transfected B16-F10 cells with targeted polymersomes loaded with luciferase and Hsp90 expression silencing siRNAs yielded 31 and 23% knockdown in target protein expression, respectively.