Application of Polymeric Micelles for Drug and Gene Delivery.

Polymeric micelles have been extensively studied because of their ability to transfer biologically active agents, such as drugs and nucleic acids [...].

Development, Characterization and Pharmacological Evaluation of Cannabidiol-Loaded Long Circulating Niosomes.

Cannabidiol (CBD) is a promising drug candidate with pleiotropic pharmacological activity, whose low aqueous solubility and unfavorable pharmacokinetics have presented obstacles to its full clinical implementation. The rational design of nanocarriers, including niosomes for CBD encapsulation, can provide a plausible approach to overcoming these limitations. The present study is focused on exploring the feasibility of copolymer-modified niosomes as platforms for systemic delivery of CBD. To confer steric stabilization, the niosomal membranes were grafted with newly synthesized amphiphilic linear or star-shaped 3- and 4-arm star-shaped copolymers based on polyglycidol (PG) and poly(ε-caprolactone) (PCL) blocks. The niosomes were prepared by film hydration method and were characterized by DLS, cryo-TEM, encapsulation efficacy, and in vitro release. Free and formulated cannabidiol were further investigated for cytotoxicity and pro-apoptotic and anti-inflammatory activities in vitro in three human tumor cell lines. The optimal formulation, based on Tween 60:Span60:Chol (3.5:3.5:3 molar ration) modified with 2.5 mol% star-shaped 3-arm copolymer, is characterized by a size of 235 nm, high encapsulation of CBD (94%), and controlled release properties. Niosomal cannabidiol retained the antineoplastic activity of the free agent, but noteworthy superior apoptogenic and inflammatory biomarker-modulating effects were established at equieffective exposure vs. the free drug. Specific alterations in key signaling molecules, implicated in programmed cell death, cancer cell biology, and inflammation, were recorded with the niosomal formulations.

Cinnamyl-Modified Polyglycidol/Poly(ε-Caprolactone) Block Copolymer Nanocarriers for Enhanced Encapsulation and Prolonged Release of Cannabidiol.

The present study describes the development of novel block copolymer nanocarriers of the phytocannabinoid cannabidiol (CBD), designed to enhance the solubility of the drug in water while achieving high encapsulation efficiency and prolonged drug release. Firstly, a well-defined amphiphilic block copolymer consisting of two outer hydrophilic polyglycidol (PG) blocks and a middle hydrophobic block of poly(ε-caprolactone) bearing pendant cinnamyl moieties (P(CyCL-co-CL)) were synthesized by the click coupling reaction of PG-monoalkyne and P(CyCL-co-CL)-diazide functional macroreagents. A non-modified polyglycidol/poly(ε-caprolactone) amphiphilic block copolymer was obtained as a referent system. Micellar carriers based on the two block copolymers were formed via the solvent evaporation method and loaded with CBD following two different protocols-loading during micelle formation and loading into preformed micelles. The key parameters/characteristics of blank and CBD-loaded micelles such as size, size distribution, zeta potential, molar mass, critical micelle concentration, morphology, and encapsulation efficiency were determined by using dynamic and static multiangle and electrophoretic light scattering, transmission electron microscopy, and atomic force microscopy. Embedding CBD into the micellar carriers affected their hydrodynamic radii to some extent, while the spherical morphology of particles was not changed. The nanoformulation based on the copolymer bearing cinnamyl moieties possessed significantly higher encapsulation efficiency and a slower rate of drug release than the non-modified copolymer. The comparative assessment of the antiproliferative effect of micellar CBD vs. the free drug against the acute myeloid leukemia-derived HL-60 cell line and Sezary Syndrome HUT-78 demonstrated that the newly developed systems have pronounced antitumor activity.

Three-Dimensional Nucleic Acid Nanostructures Based on Self-Assembled Polymer-Oligonucleotide Conjugates of Comblike and Coil-Comb Chain Architectures.

Spherical nucleic acids have emerged as a class of nanostructures, exhibiting a wide variety of properties, distinctly different from those of linear nucleic acids, and a plethora of applications in therapeutics and diagnostics. Herein, we report on preparation of 3D nucleic acid nanostructures, prepared by self-assembly of polymer-oligonucleotide conjugates. The latter are obtained by grafting multiple alkyne-functionalized oligonucleotide strands onto azide-modified homo-, block, and random (co)polymers of chloromethylstyrene via initiator-free click coupling chemistry to form conjugates of comblike and coil-comb chain architectures. The resulting conjugates are amphiphilic and form stable nanosized supramolecular structures in aqueous solution. The nanoconstructs are thoroughly investigated and a number of physical characteristics, in particular, molar mass, size, aggregation number, zeta potential, material density, number of oligonucleotide strands per particle, grafting density, and their relation to hallmark properties of spherical nucleic acids - biocompatibility, resistance against DNase I, cellular uptake without the need for transfection agents - are determined.

Influence of DNA Type on the Physicochemical and Biological Properties of Polyplexes Based on Star Polymers Bearing Different Amino Functionalities.

The interactions of two star polymers based on poly (2-(dimethylamino)ethyl methacrylate) with different types of nucleic acids are investigated. The star polymers differ only in their functionality to bear protonable amino or permanently charged quaternary ammonium groups, while DNAs of different molar masses, lengths and topologies are used. The main physicochemical parameters of the resulting polyplexes are determined. The influence of the polymer' functionality and length and topology of the DNA on the structure and properties of the polyelectrolyte complexes is established. The quaternized polymer is characterized by a high binding affinity to DNA and formed strongly positively charged, compact and tight polyplexes. The parent, non-quaternized polymer exhibits an enhanced buffering capacity and weakened polymer/DNA interactions, particularly upon the addition of NaCl, resulting in the formation of less compact and tight polyplexes. The cytotoxic evaluation of the systems indicates that they are sparing with respect to the cell lines studied including osteosarcoma, osteoblast and human adipose-derived mesenchymal stem cells and exhibit good biocompatibility. Transfection experiments reveal that the non-quaternized polymer is effective at transferring DNA into cells, which is attributed to its high buffering capacity, facilitating the endo-lysosomal escape of the polyplex, the loose structure of the latter one and weakened polymer/DNA interactions, benefitting the DNA release.

Original Synthesis of a Nucleolipid for Preparation of Vesicular Spherical Nucleic Acids.

Spherical nucleic acids (SNAs)-nanostructures, consisting of a nanoparticle core densely functionalized with a shell of short oligonucleotide strands-are a rapidly emerging class of nanoparticle-based therapeutics with unique properties and specific applications as drug and nucleic acid delivery and gene regulation materials. In this contribution, we report on the preparation of hollow SNA nanoconstructs by co-assembly of an originally synthesized nucleolipid-a hybrid biomacromolecule, composed of a lipidic residue, covalently linked to a DNA oligonucleotide strand-with other lipids. The nucleolipid was synthesized via a click chemistry approach employing initiator-free, UV light-induced thiol-ene coupling of appropriately functionalized intermediates, performed in mild conditions using a custom-made UV light-emitting device. The SNA nanoconstructs were of a vesicular structure consisting of a self-closed bilayer membrane in which the nucleolipid was intercalated via its lipid-mimetic residue. They were in the lower nanometer size range, moderately negatively charged, and were found to carry thousands of oligonucleotide strands per particle, corresponding to a grafting density comparable to that of other SNA structures. The surface density of the strands on the bilayer implied that they adopted an unextended conformation. We demonstrated that preformed vesicular structures could be successfully loaded with either hydrophilic or hydrophobic dyes.

Nucleic acid-based supramolecular structures: vesicular spherical nucleic acids from a non-phospholipid nucleolipid.

Vesicular spherical nucleic acids are dynamic nucleic acid-based supramolecular structures that are held together via non-covalent bonds. They have promising applications as drug and nucleic acid delivery materials, diagnostic and imaging tools and platforms for development of various therapeutic schemes. In this contribution, we report on vesicular spherical nucleic acids, constructed from a non-phospholipid nucleolipid - an original hybrid biomacromolecule, composed of a hydrophobic residue, resembling that of the naturally occurring phospholipids, and a DNA oligonucleotide strand. The nucleolipid was synthesized by coupling of dibenzocyclooctyne-functionalized oligonucleotide and azidated 1,3-dihexadecyloxy-propane-2-ol via an azide-alkyne click reaction. In aqueous solution it spontaneously self-associated into nanosized supramolecular structures, identified as unilamellar vesicles composed of a self-closed interdigitated bilayer. Vesicular structures were also formed upon intercalation of the nucleolipid via its lipid-mimetic residue in the phospholipid bilayer membrane of liposomes prepared from readily available and FDA-approved lipids (1,2-dipalmitoyl-rac-glycero-3-phosphocholine and cholesterol). The vesicular structures are thoroughly investigated by light scattering (dynamic, static, and electrophoretic) and cryogenic TEM and the physical characteristics, in particular, number of strands per particle, grafting density, and conformation of the strands, were compared to those of reference spherical nucleic acids. Finally, the vesicular structures were shown to exhibit cellular internalization with no need of transfection agents and enhanced colloidal and nuclease stability.

Hollow spherical nucleic acid structures based on polymer-coated phospholipid vesicles.

A feasible one pot synthesis of hollow spherical nucleic acids (SNAs) using phospholipid liposomes is reported. These constructs are synthesized in a chemically straightforward process involving formation of unilamellar liposomes, coating the liposomes with a thin cross-linked polymeric layer, and grafting the latter with short (about 20 bases) DNA oligonucleotide strands. They consist of vesicular cores, composed of readily available phospholipid (1,2-dipalmitoyl-sn-glycero-phosphocholine), whereas the strands are deliberately arranged on the surface of the vesicular entities. The initial vesicular structure and morphology are preserved during the coating and grafting reactions. The novel hollow/vesicular SNAs are characterized with a hydrodynamic radius and radius of gyration of 78.3 and 88.5 nm, respectively, and moderately negative (-14.2 mV) ζ potential. They carry thousands (5868) of oligonucleotide strands per vesicle, which are not strongly radially oriented and adopt an unextended conformation as anticipated from the smaller value of the grafting density compared to the critical grafting density at the transition to brush conformation. The constructs are practically devoid of toxicity and exhibit high binding affinity to complementary nucleic acids. Unlike any other nucleic acid structural motif, they cross the cell membrane and enter cells without the need of transfection agents.

Formulation and Evaluation of Hybrid Niosomal In Situ Gel for Intravesical Co-Delivery of Curcumin and Gentamicin Sulfate.

The current study describes the elaboration of a hybrid drug delivery platform for an intravesical application based on curcumin/gentamicin sulfate simultaneously loaded niosomes incorporated into thermosensitive in situ gels. Series of niosomes were elaborated via the thin film hydration method, evaluating the impact of non-ionic surfactants', cholesterol's, and curcumin's concentration. The formulation composed of equimolar ratio of Span 60, Tween 60, and 30 mol% cholesterol was selected as the optimal composition, due to the high entrapment efficiency values obtained for both drugs, and appropriate physicochemical parameters (morphology, size, PDI, and zeta potential), therefore, was further incorporated into Poloxamers (407/188) and Poloxamers and chitosan based in situ gels. The developed hybrid systems were characterized with sol to gel transition in the physiological range, suitable rheological and gelling characteristics. In addition, the formed gel structure at physiological temperatures determines the retarded dissolution of both drugs (vs. niosomal suspension) and sustained release profile. The conducted microbial studies of selected niosomal in situ gels revealed the occurrence of a synergetic effect of the two compounds when simultaneously loaded. The findings indicate that the elaborated thermosensitive niosomal in situ gels can be considered as a feasible platform for intravesical drug delivery.

PEG-Modified tert-Octylcalix[8]arenes as Drug Delivery Nanocarriers of Silibinin.

The hepatoprotective properties of silibinin, as well its therapeutic potential as an anticancer and chemo-preventive agent, have failed to progress towards clinical development and commercialization due to this material's unfavorable pharmacokinetics and physicochemical properties, low aqueous solubility, and chemical instability. The present contribution is focused on the feasibility of using PEGylated calixarene, in particular polyoxyethylene-derivatized tert-octylcalix[8]arene, to prepare various platforms for the delivery of silibinin, such as inclusion complexes and supramolecular aggregates thereof. The inclusion complex is characterized by various instrumental methods. At concentrations exceeding the critical micellization concentration of PEGylated calixarene, the tremendous solubility increment of silibinin is attributed to the additional solubilization and hydrophobic non-covalent interactions of the drug with supramolecular aggregates. PEG-modified tert-octylcalix[8]arenes, used as drug delivery carriers for silibinin, were additionally investigated for cytotoxicity against human tumor cell lines.

Unprecedented formation of sterically stabilized phospholipid liposomes of cuboidal morphology.

Sterically stabilized phospholipid liposomes of unprecedented cuboid morphology are formed upon introduction in the bilayer membrane of original polymers, based on polyglycidol bearing a lipid-mimetic residue. Strong hydrogen bonding in the polyglycidol sublayers creates attractive forces, which, facilitated by fluidization of the membrane, bring about the flattening of the bilayers and the formation of cuboid vesicles.

Functional Polyglycidol-Based Block Copolymers for DNA Complexation.

Gene therapy is an attractive therapeutic method for the treatment of genetic disorders for which the efficient delivery of nucleic acids into a target cell is critical. The present study is aimed at evaluating the potential of copolymers based on linear polyglycidol to act as carriers of nucleic acids. Functional copolymers with linear polyglycidol as a non-ionic hydrophilic block and a second block bearing amine hydrochloride pendant groups were prepared using previously synthesized poly(allyl glycidyl ether)-b-polyglycidol block copolymers as precursors. The amine functionalities were introduced via highly efficient radical addition of 2-aminoethanethiol hydrochloride to the alkene side groups. The modified copolymers formed loose aggregates with strongly positive surface charge in aqueous media, stabilized by the presence of dodecyl residues at the end of the copolymer structures and the hydrogen-bonding interactions in polyglycidol segments. The copolymer aggregates were able to condense DNA into stable and compact nanosized polyplex particles through electrostatic interactions. The copolymers and the corresponding polyplexes showed low to moderate cytotoxicity on a panel of human cancer cell lines. The cell internalization evaluation demonstrated the capability of the polyplexes to successfully deliver DNA into the cancer cells.

Cationic (Co)polymers Based on N-Substituted Polyacrylamides as Carriers of Bio-macromolecules: Polyplexes, Micelleplexes, and Spherical Nucleic Acidlike Structures.

Novel N-substituted polyacrylamides bearing a cycle with two tertiary amines, poly(4-methyl-piperazin-1-yl)-propenone (PMPP) and its block copolymers with polylactide (PMPP-b-PLA), are synthesized and characterized. The homopolymers are water-soluble, whereas the block copolymers self-assemble in aqueous solution into a small size (Rh around 30 nm), are narrowly distributed, and exhibit core-shell micelles with good colloidal stability. Both the homopolymers and copolymer micelles are positively charged (ζ-potentials in the 13.8-17.6 mV range), which are employed for formation of electrostatic complexes with oppositely charged DNA. Complexes (polyplexes, micelleplexes, and spherical nucleic acidlike structures) in a wide range of N/P (amino to phosphate groups) ratios are prepared with short (115 bp) and long (2000 bp) DNA. The behavior and physicochemical properties of the resulting nanocarriers of DNA are strongly dependent on the polymer/polymer micelles' characteristics and the DNA chain length. All systems exhibit low cytotoxicity and good cellular uptake ability and show promise for gene delivery and regulation.

Physicochemical Properties and Biological Performance of Polymethacrylate Based Gene Delivery Vector Systems: Influence of Amino Functionalities.

Physicochemical characteristics and biological performance of polyplexes based on two identical copolymers bearing tertiary amino or quaternary ammonium groups are evaluated and compared. Poly(2-(dimethylamino)ethyl methacrylate)-b-poly(oligo(ethylene glycol) methyl ether methacrylate) block copolymer (PDMAEMA-b-POEGMA) is synthesized by reversible addition fragmentation chain transfer polymerization. The tertiary amines of PDMAEMA are converted to quaternary ammonium groups by quaternization with methyl iodide. The two copolymers spontaneously formed well-defined polyplexes with DNA. The size, zeta potential, molar mass, aggregation number, and morphology of the polyplex particles are determined. The parent PDMAEMA-b-POEGMA exhibits larger buffering capacity, whereas the corresponding quaternized copolymer (QPDMAEMA-b-POEGMA) displays stronger binding affinity to DNA, yielding invariably larger in size and molar mass particles bearing greater number of DNA molecules per particle. Experiments revealed that QPDMAEMA-b-POEGMA is more effective in transfecting pEGFP-N1 than the parent copolymer, attributed to the larger size, molar mass, and DNA cargo, as well as to the effective cellular traffic, which dominated over the enhanced ability for endo-lysosomal escape of PDMAEMA-b-POEGMA.

Development and evaluation of doxycycline niosomal thermoresponsive in situ gel for ophthalmic delivery.

The present study was focused on the development of doxycycline niosomal thermosensitive in situ gel for ophthalmic application. For this purpose, in situ gel formulations based on Poloxamer 407 alone and in combination with hydroxypropyl methylcellulose were prepared by cold method and evaluated in terms of sol-gel transition temperature, gelling time and capacity. The addition of hydroxypropyl methylcellulose to the composition led to decrease in the phase transition temperature of the systems. Conversely, the inclusion of doxycycline niosomes to the formulations didn''t have a significant influence on their gelling and rheological properties. Doxycycline niosomal in situ gel based on 15%w/w Poloxamer and 1.5% w/w hydroxypropyl methylcellulose was characterized with gelation temperature of 34 °C, appropriate for ophthalmic application, pseudoplastic flow behavior and very good physical stability. In vitro release studies indicated slower and sustained doxycycline release from the developed in situ gel as compared to niosomes. The conducted microbiological studies revealed its enhanced antibacterial activity with respect to doxycycline solution and doxycycline in situ gel. The obtained results indicate that the elaborated niosomal in situ gel may serve as a promising system for ophthalmic delivery of doxycycline, ensuring sufficient therapeutic concentration and sustained drug release.

Thermoresponsive Polyoxazolines as Vectors for Transfection of Nucleic Acids.

Poly(2-oxazoline)s (POx) are an attractive platform for the development of non-viral gene delivery systems. The combination of POx moieties, exhibiting excellent biocompatibility, with DNA-binding polyethyleneimine (PEI) moieties into a single copolymer chain is a promising approach to balance toxicity and transfection efficiency. The versatility of POx in terms of type of substituent, copolymer composition, degree of polymerization, degree of hydrolysis, and chain architecture, as well as the introduction of stimuli-responsive properties, provides opportunities to finely tune the copolymer characteristics and physicochemical properties of the polyplexes to increase the biological performance. An overview of the current state of research in the POx-PEI-based gene delivery systems focusing particularly on thermosensitive POx is presented in this paper.

Safety assessment of a newly synthesized copolymer for micellar delivery of hydrophobic caffeic acid phenethyl ester.

Caffeic acid phenethyl ester (CAPE), a major pharmacologically active component of poplar type propolis, is known for its proapoptotic, anti-inflammatory, antioxidant, antiviral, and enzyme inhibiting activities. The aim of this study was to perform an in vitro and in vivo safety assessment of a micellar system based on a newly synthesized copolymer, consisting of polyglycidol and poly(allyl glycidyl ether) (C12-PAGE-PG) as a drug delivery platform for CAPE. The in vitro studies on HepG2 and L929 cells by MTT and LDH assays after treatment with the empty and CAPE-loaded micelles showed no cytotoxic effects of the empty micelles and retained cytotoxic activity of CAPE loaded in the micelles. No hemolysis or stimulation of mouse lymphocytes or macrophages was observed in vitro. In vivo hematological, biochemical, and histological assays on rats, treated with the empty (2580 and 5160 µg/kg) or CAPE-loaded (375 and 750 µg CAPE/kg) micelles did not reveal pathological changes of any of the parameters assayed after 14-days' treatment. In conclusion, initial toxicological data characterize C12-PAGE-PG as a non-toxic and promising copolymer for development of micellar drug delivery systems, particularly for a hydrophobic active substance as CAPE.

One-pot synthesis of oligonucleotide-grafted polymeric nanoparticles.

A feasible one-pot approach for constructing oligonucleotide-grafted polymeric nanoparticles is reported. The approach involves formation of mesoglobules from a thermoresponsive polymer, coating of the mesoglobules with a cross-linked polymeric shell, and grafting the latter with oligonucleotide strands. Dynamic and static light scattering are used to parameterize the novel constructs. They are relatively large structures with hydrodynamic radii and molar masses reaching 200 nm and 150.0 × 106 g mol-1, respectively. The oligonucleotide-grafted polymeric nanoparticles are of spherical morphology and moderately negative (-12.4 to -19.1 mV) ζ potential as revealed by AFM, TEM, and electrophoretic light scattering. In accordance with their large size, they are found to carry thousands of oligonucleotide strands per particle. The novel constructs are thermoresponsive. They undergo reversible collapse upon heating and swelling upon cooling, which is associated with changes in the grafting density and, hence, the conformation of the oligonucleotide strands from unextended at room temperature to a more extended one at elevated temperatures. The versatility of the approach is demonstrated by varying the type of the cross-linked shell and content of the oligonucleotide strands and, hence, the grafting density. Appropriate diversification and modifications are suggested as well.

Design and in vitro evaluation of doxycycline hyclate niosomes as a potential ocular delivery system.

Niosomes have been considered as promising nanoscale carriers for ocular drug delivery, since they have been shown to increase the bioavailability of various drugs and to improve their efficacy. The main objective of this study was to prepare and characterize niosomes for ocular delivery of doxycycline hyclate. Niosomes were prepared using various surfactants (namely Span 20, Span 60, Span 80, Tween 60) and cholesterol in different molar ratios, using the thin film hydration method followed by multiple membrane extrusion or the reverse-phase evaporation method. In our hands highest entrapment efficiency was encountered with the formulation composed of Span 60 and cholesterol, prepared by the reverse phase evaporation method. Transmission electron microscopy and dynamic light scattering were used to assess the morphology, size and size distribution paterns of prepared niosomes. In vitro release studies showed sustained release of doxycycline from niosomes. After 2 months of storage at 4 °C the doxycycline-loaded niosomes remained physically stable in terms of encapsulation efficiency and particle size. The performed Draize test revealed that the prepared niosomes were well tolerated by the eye. Taken together our findings indicate that niosomes could be considered as a plausible drug delivery platform for for ophthalmic application of doxycycline.

Self-Assembly of a Thermally Responsive Double-Hydrophilic Copolymer in Ethanol-Water Mixtures: The Effect of Preferential Adsorption and Co-Nonsolvency.

Lower alcohols can induce a combined collapse-swelling de-mixing transition (lower critical solution temperature (LCST)-type co-nonsolvency) in aqueous solutions of poly( N-isopropylacrylamide) (PNIPAM) by interacting with the polymer's amide groups. This interaction results in an increase of the total surface area of hydrophobic sites and destabilizes the chains. Here, we make use of this phenomenon to drive the counterintuitive self-assembly of a PNIPAM-containing double-hydrophilic graft copolymer in water-ethanol mixtures at T ≪ LCST. Rheological frequency sweeps are used to quantify the distinct solvation states of PNIPAM at various temperatures and ethanol concentrations. The energy stored through elastic deformation at the de-mixing transition is simply related to the solvent binding. We find that the storage modulus decreases progressively, but nonlinearly with ethanol concentration, which evidences a preferential solvation pattern. Analogously, through a combination of dynamic light scattering and transmission electron microscope analyses, we demonstrate that a low-temperature structure variation takes place by adding ethanol following a similar solvent-content morphology dependent model.