Barcode lipids for absolute quantitation of liposomes in ocular tissues.

Lipid-based drug formulations are promising systems for improving delivery of drugs to ocular tissues, such as retina. To develop lipid-based systems further, an improved understanding of their pharmacokinetics is required, but high-quality in vivo experiments require a large number of animals, raising ethical and economic questions. In order to expedite in vivo kinetic testing of lipid-based systems, we propose a barcode approach that is based on barcoding liposomes with non-endogenous lipids. We developed and evaluated a liquid-chromatography-mass spectrometry method to quantify many liposomes simultaneously in aqueous humor, vitreous, and neural retina at higher than ±20% precision and accuracy. Furthermore, we showed in vivo suitability of the method in pharmacokinetic evaluation of six different liposomes after their simultaneous injection into the rat vitreal cavity. We calculated pharmacokinetic parameters in vitreous and aqueous humor, quantified liposome concentrations in the retina, and quantitated retinal distribution of the liposomes in the rats. Compared to individual injections of the liposome formulations, the barcode-based study design enabled reduction of animal numbers from 72 to 12. We believe that the proposed approach is reliable and will reduce and refine ocular pharmacokinetic experiments with liposomes and other lipid-based systems.

Dual drug loaded polypeptide delivery systems for cancer therapy.

The present study was aimed to prepare and examine in vitro novel dual-drug loaded delivery systems. Biodegradable nanoparticles based on poly(L-glutamic acid-co-D-phenylalanine) were used as nanocarriers for encapsulation of two drugs from the paclitaxel, irinotecan, and doxorubicin series. The developed delivery systems were characterised with hydrodynamic diameters less than 300 nm (PDI < 0.3). High encapsulation efficiencies (≥75%) were achieved for all single- and dual-drug formulations. The release studies showed faster release at acidic pH, with the release rate decreasing over time. The release patterns of the co-encapsulated forms of substances differed from those of the separately encapsulated drugs, suggesting differences in drug-polymer interactions. The joint action of encapsulated drugs was analysed using the colon cancer cells, both for the dual-drug delivery sytems and a mixture of single-drug formulations. The encapsulated forms of the drug combinations demonstrated comparable efficacy to the free forms, with the encapsulation enhancing solubility of the hydrophobic drug paclitaxel.

Synthesis and Characterization of Nanoparticle-Based Dexamethasone-Polypeptide Conjugates as Potential Intravitreal Delivery Systems.

The use of dexamethasone for eye disease treatment is limited by its low solubility, bioavailability, and rapid elimination when applied topically. The covalent conjugation of dexamethasone with polymeric carriers is a promising strategy to overcome existing drawbacks. In this work, amphiphilic polypeptides capable of self-assembly into nanoparticles were proposed as potential delivery systems for intravitreal delivery. The nanoparticles were prepared and characterized using poly(L-glutamic acid-co-D-phenylalanine) and poly(L-lysine-co-D/L-phenylalanine) as well as poly(L-lysine-co-D/L-phenylalanine) covered with heparin. The critical association concentration for the polypeptides obtained was in the 4.2-9.4 µg/mL range. The hydrodynamic size of the formed nanoparticles was between 90 and 210 nm, and they had an index of polydispersity between 0.08 and 0.27 and an absolute zeta-potential value between 20 and 45 mV. The ability of nanoparticles to migrate in the vitreous humor was examined using intact porcine vitreous. Conjugation of DEX with polypeptides was performed by additional succinylation of DEX and activation of carboxyl groups introduced to react with primary amines in polypeptides. The structures of all intermediate and final compounds were verified by 1H NMR spectroscopy. The amount of conjugated DEX can be varied from 6 to 220 µg/mg of polymer. The hydrodynamic diameter of the nanoparticle-based conjugates was increased to 200-370 nm, depending on the polymer sample and drug loading. The release of DEX from the conjugates due to hydrolysis of the ester bond between DEX and the succinyl moiety was studied both in a buffer medium and a vitreous/buffer mixture (50/50, v/v). As expected, the release in the vitreous medium was faster. However, the release rate could be controlled in the range of 96-192 h by varying the polymer composition. In addition, several mathematical models were used to assess the release profiles and figure out how DEX is released.

Biocompatible Nanoparticles Based on Amphiphilic Random Polypeptides and Glycopolymers as Drug Delivery Systems.

In this research, the development and investigation of novel nanoobjects based on biodegradable random polypeptides and synthetic non-degradable glycopolymer poly(2-deoxy-2-methacrylamido-d-glucose) were proposed as drug delivery systems. Two different approaches have been applied for preparation of such nanomaterials. The first one includes the synthesis of block-random copolymers consisting of polypeptide and glycopolymer and capable of self-assembly into polymer particles. The synthesis of copolymers was performed using sequential reversible addition-fragmentation chain transfer (RAFT) and ring-opening polymerization (ROP) techniques. Amphiphilic poly(2-deoxy-2-methacrylamido-d-glucose)-b-poly(l-lysine-co-l-phenylalanine) (PMAG-b-P(Lys-co-Phe)) copolymers were then used for preparation of self-assembled nanoparticles. Another approach for the formation of polypeptide-glycopolymer particles was based on the post-modification of preformed polypeptide particles with an oxidized glycopolymer. The conjugation of the polysaccharide on the surface of the particles was achieved by the interaction of the aldehyde groups of the oxidized glycopolymer with the amino groups of the polymer on particle surface, followed by the reduction of the formed Schiff base with sodium borohydride. A comparative study of polymer nanoparticles developed with its cationic analogues based on random P(Lys-co-d-Phe), as well as an anionic one-P(Lys-co-d-Phe) covered with heparin--was carried out. In vitro antitumor activity of novel paclitaxel-loaded PMAG-b-P(Lys-co-Phe)-based particles towards A549 (human lung carcinoma) and MCF-7 (human breast adenocarcinoma) cells was comparable to the commercially available Paclitaxel-LANS.

Self-Assembled Nanoparticles Based on Block-Copolymers of Poly(2-Deoxy-2-methacrylamido-d-glucose)/Poly(N-Vinyl Succinamic Acid) with Poly(O-Cholesteryl Methacrylate) for Delivery of Hydrophobic Drugs.

The self-assembly of amphiphilic block-copolymers is a convenient way to obtain soft nanomaterials of different morphology and scale. In turn, the use of a biomimetic approach makes it possible to synthesize polymers with fragments similar to natural macromolecules but more resistant to biodegradation. In this study, we synthesized the novel bio-inspired amphiphilic block-copolymers consisting of poly(N-methacrylamido-d-glucose) or poly(N-vinyl succinamic acid) as a hydrophilic fragment and poly(O-cholesteryl methacrylate) as a hydrophobic fragment. Block-copolymers were synthesized by radical addition-fragmentation chain-transfer (RAFT) polymerization using dithiobenzoate or trithiocarbonate chain-transfer agent depending on the first monomer, further forming the hydrophilic block. Both homopolymers and copolymers were characterized by 1H NMR and Fourier transform infrared spectroscopy, as well as thermogravimetric analysis. The obtained copolymers had low dispersity (1.05-1.37) and molecular weights in the range of ~13,000-32,000. The amphiphilic copolymers demonstrated enhanced thermal stability in comparison with hydrophilic precursors. According to dynamic light scattering and nanoparticle tracking analysis, the obtained amphiphilic copolymers were able to self-assemble in aqueous media into nanoparticles with a hydrodynamic diameter of approximately 200 nm. An investigation of nanoparticles by transmission electron microscopy revealed their spherical shape. The obtained nanoparticles did not demonstrate cytotoxicity against human embryonic kidney (HEK293) and bronchial epithelial (BEAS-2B) cells, and they were characterized by a low uptake by macrophages in vitro. Paclitaxel loaded into the developed polymer nanoparticles retained biological activity against lung adenocarcinoma epithelial cells (A549).

Synthesis and Characterization of Macroinitiators Based on Polyorganophosphazenes for the Ring Opening Polymerization of N-Carboxyanhydrides.

Among the various biocompatible amphiphilic copolymers, biodegradable ones are the most promising for the preparation of drug delivery systems since they are destroyed under physiological conditions, that, as a rule, reduce toxicity and provide controlled release of the drug. Hybrid graft-copolymers consisting of the main inorganic polyphosphazene chain and polypeptide side chains are of considerable interest for the development of delivery systems with a controlled degradation rate, since the main and side chains will have different degradation mechanisms (chemical and enzymatic hydrolysis, respectively). Variable particle degradation rate, controlled by the adjusting the composition of substituents, will allow selective delivery in vivo and controlled drug release. The present work proposes the preparation of biodegradable macroinitiators based on polyorganophosphazenes for the synthesis of hybrid copolymers. Synthesis of novel biodegradable macroinitiators based on polyorganophosphazenes was performed via macromolecular substitution of a polydichlorophosphazene chain with the sodium alcoholates, amines and amino acids. The composition of copolymers obtained was calculated using NMR. These polyorganophosphazenes bearing primary amino groups can be considered as convenient macroinitiators for the polymerization of NCA of α-amino acids in order to prepare hybrid copolymers polyphosphazene-graft-polypeptide. The developed macroinitiators were amphiphilic and self-assembled in the aqueous media into nanoparticles. Furthermore, the ability to encapsulate and release a model substance was demonstrated. In addition, the in vitro cytotoxicity of synthesized polymers was evaluated using two cell lines.

Polypeptide Self-Assembled Nanoparticles as Delivery Systems for Polymyxins B and E.

Polymyxins are peptide antibiotics that are highly efficient against many multidrug resistant pathogens. However, the poor stability of polymyxins in the bloodstream requires the administration of high drug doses that, in turn, can lead to polymyxin toxicity. Consequently, different delivery systems have been considered for polymyxins to overcome these obstacles. In this work, we report the development of polymyxin delivery systems based on nanoparticles obtained from the self-assembly of amphiphilic random poly(l-glutamic acid-co-d-phenylalanine). These P(Glu-co-dPhe) nanoparticles were characterized in terms of their size, surface charge, stability, cytotoxicity, and uptake by macrophages. The encapsulation efficiency and drug loading into P(Glu-co-dPhe) nanoparticles were determined for both polymyxin B and E. The release kinetics of polymyxins B and E from nanoformulations was studied and compared in buffer solution and human blood plasma. The release mechanisms were analyzed using a number of mathematical models. The minimal inhibitory concentrations of the nanoformulations were established and compared with those determined for the free antibiotics.

Bio-Inspired Amphiphilic Block-Copolymers Based on Synthetic Glycopolymer and Poly(Amino Acid) as Potential Drug Delivery Systems.

In this work, a method to prepare hybrid amphiphilic block copolymers consisting of biocompatible synthetic glycopolymer with non-degradable backbone and biodegradable poly(amino acid) (PAA) was developed. The glycopolymer, poly(2-deoxy-2-methacrylamido-D-glucose) (PMAG), was synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Two methods for modifying the terminal dithiobenzoate-group of PMAG was investigated to obtain the macroinitiator bearing a primary aliphatic amino group, which is required for ring-opening polymerization of N-carboxyanhydrides of hydrophobic α-amino acids. The synthesized amphiphilic block copolymers were carefully analyzed using a set of different physico-chemical methods to establish their composition and molecular weight. The developed amphiphilic copolymers tended to self-assemble in nanoparticles of different morphology that depended on the nature of the hydrophobic amino acid present in the copolymer. The hydrodynamic diameter, morphology, and cytotoxicity of polymer particles based on PMAG-b-PAA were evaluated using dynamic light scattering (DLS) and transmission electron microscopy (TEM), as well as CellTiter-Blue (CTB) assay, respectively. The redox-responsive properties of nanoparticles were evaluated in the presence of glutathione taken at different concentrations. Moreover, the encapsulation of paclitaxel into PMAG-b-PAA particles and their cytotoxicity on human lung carcinoma cells (A549) and human breast adenocarcinoma cells (MCF-7) were studied.

Amphiphilic Polypeptides for VEGF siRNA Delivery into Retinal Epithelial Cells.

Polyethyleneimine, poly-L-lysine, chitosan and some others cationic polymers have been thoroughly studied as nucleic acid delivery systems in gene therapy. However, the drug release from these systems proceeds at a very low rate due to extremely high binding between a carrier and gene material. To reduce these interactions and to enhance drug release, we developed a set of amphiphilic polypeptides containing positively and negatively charged amino acids as well as a hydrophobic one. The copolymers obtained were characterized by size-exclusion chromatography, static light scattering, HPLC amino acid analysis and 1HNMR spectroscopy. All copolymers formed particles due to a self-assembly in aqueous media. Depending on polypeptide composition, the formation of particles with hydrodynamic diameters from 180 to 900 nm was observed. Stability of polymer particles, loading and release efficiency were carefully studied. Cellular uptake of the particles was efficient and their cytotoxicity was negligible. The application of polymer carriers, containing siRNA, to vascular endothelial growth factor (VEGF-A165) silencing of ARPE-19 cells was successful. The gene silencing was confirmed by suppression of both messenger RNA and protein expression.

Novel Formulations of C-Peptide with Long-Acting Therapeutic Potential for Treatment of Diabetic Complications.

The development and application of novel nanospheres based on cationic and anionic random amphiphilic polypeptides with prolonged stability were proposed. The random copolymers, e.g., poly(l-lysine-co-d-phenylalanine) (P(Lys-co-dPhe)) and poly(l-glutamic acid-co-d-phenylalanine) (P(Glu-co-dPhe)), with different amount of hydrophilic and hydrophobic monomers were synthesized. The polypeptides obtained were able to self-assemble into nanospheres. Such characteristics as size, PDI and ζ-potential of the nanospheres were determined, as well as their dependence on pH was also studied. Additionally, the investigation of their biodegradability and cytotoxicity was performed. The prolonged stability of nanospheres was achieved via introduction of d-amino acids into the polypeptide structure. The cytotoxicity of nanospheres obtained was tested using HEK-293 cells. It was proved that no cytotoxicity up to the concentration of 500 µg/mL was observed. C-peptide delivery systems were realized in two ways: (1) peptide immobilization on the surface of P(Glu-co-dPhe) nanospheres; and (2) peptide encapsulation into P(Lys-co-dPhe) systems. The immobilization capacity and the dependence of C-peptide encapsulation efficiency, as well as maximal loading capacity, on initial drug concentration was studied. The kinetic of drug release was studied at model physiological conditions. Novel formulations of a long-acting C-peptide exhibited their effect ex vivo by increasing activity of erythrocyte Na⁺/K⁺-adenosine triphosphatase.

Amphiphilic polypeptides with prolonged enzymatic stability for the preparation of self-assembled nanobiomaterials.

Due to their excellent biocompatibility and biodegradability, polypeptides have emerged as versatile bio-inspired scaffolds for the preparation of artificial biomaterials. In order to create self-assembled polypeptide nanoparticles with enhanced stability towards enzymatic degradation, we synthesized a series of random and block polypeptides based on lysine and α-aminoisobutyric acid (Aib) by the ring-opening polymerization of N-carboxyanhydrides (ROP NCA) of the corresponding amino acids. A conformational analysis carried out by means of FT-IR absorption and CD spectroscopies revealed a noticeable difference between random and block copolymers. In turn, the spatial organization of the polypeptide chains induced the formation of nanostructures of different types. The block copolymers self-assembled in vesicle-like structures, whereas polypeptides with randomly distributed monomers formed micelles. In contrast with the polymers with only natural amino acids, all nanoparticles based on Aib/Lys polypeptides showed strong resistance to proteolytic cleavage. The cytotoxicity and the kinetics of the cellular uptake of the prepared nanostructures were also studied. The results obtained could not only contribute to the understanding of long Aib polypeptide folding and self-assembling, but also pave the way to the design of nanomaterials with finely tuned properties in the fields of drug delivery and tissue engineering.

Nanotraps with biomimetic surface as decoys for chemokines.

A creation of nanotraps that could selectively recognize the chemotactic mediators of leukocyte adhesion and eliminate them from the bloodstream and tissue intercellular matrix is a promising approach for the treatment of various inflammatory and autoimmune diseases. We designed nanotraps as artificial decoy receptors based on poly(lactic acid) (PLA) nanoparticles covered by heparin and bearing on the surface two fragments of CCR5 receptor (N-terminal domain, Nt, and second extracellular loop, ECL2), responsible for chemokine binding. In order to attach Nt and ECL2 to the heparin shell, the corresponding peptides were modified with N- and/or C-terminal oligolysines. The presence of the nanotraps in the cell medium completely eliminated the activating effect of a CCR5 ligand, chemokine Rantes, while strongly decreasing the adhesion of monocytes to the human endothelial cells. We found that the modified ECL2 alone was also able to prevent monocyte adhesion, thus acting as a decoy receptor itself.

Preparation, Characterization, and Biological Evaluation of Poly(Glutamic Acid)-b-Polyphenylalanine Polymersomes.

Different types of amphiphilic macromolecular structures have been developed within recent decades to prepare the polymer particles considered as drug delivery systems. In the present research the series of amphiphilic block-copolymers containing poly(glutamatic acid) as hydrophilic, and polyphenylalanine as hydrophobic blocks was synthesized and characterized. Molecular weights for homo- and copolymers were determined by gel-permeation chromatography (GPC) and amino acid analysis, respectively. The copolymers obtained were applied for preparation of polymer particles. The specific morphology of prepared polymerosomes was proved using transmission electron microscopy (TEM). The influence on particle size of polymer concentration and pH used for self-assembly, as well as on the length of hydrophobic and hydrophilic blocks of applied copolymers, was studied by dynamic light scattering (DLS). Depending on different experimental conditions, the formation of nanoparticles with sizes from 60 to 350 nm was observed. The surface of polymersomes was modified with model protein (enzyme). No loss in biocatalytic activity was detected. Additionally, the process of encapsulation of model dyes was developed and the possibility of intracellular delivery of the dye-loaded nanoparticles was proved. Thus, the nanoparticles discussed can be considered for the creation of modern drug delivery systems.