Cyclodextrin-modified polyethylenimine polymers for gene delivery

Linear and branched poly(ethylenimines), lPEI and bPEI, respectively, grafted with beta-cyclodextrin are prepared to give CD-lPEI and CD-bPEI, respectively, and are investigated as in vitro and in vivo nonviral gene delivery agents. The in vitro toxicity and transfection efficiency are sensitive to the level of cyclodextrin grafting. The cyclodextrin-containing polycations, when combined with adamantane-poly(ethylene glycol) (AD-PEG) conjugates, form particles that are stable at physiological salt concentrations. PEGylated CD-lPEI-based particles give in vitro gene expression equal to or greater than lPEI as measured by the percentage of EGFP expressing cells. Tail vein injections into mice of 120 microg of plasmid DNA formulated with CD-lPEI and AD-PEG do not reveal observable toxicities, and both nucleic acid accumulation and expression are observed in liver.     

Molecularly imprinted polymers for drug delivery

Molecular imprinting technology has an enormous potential for creating satisfactory drug dosage forms. Although its application in this field is just at an incipient stage, the use of MIPs in the design of new drug delivery systems (DDS) and devices useful in closely related fields, such as diagnostic sensors, is receiving increasing attention. Examples of MIP-based DDS can be found for the three main approaches developed to control the moment at which delivery should begin and/or the drug release rate, i.e. rate-programmed, activation-modulated, or feedback-regulated drug delivery. The utility of these systems for administering drugs by different routes (e.g. oral, ocular or transdermal) or trapping undesired substances under in vivo conditions is discussed. This review seeks to highlight the more remarkable advantages of the imprinting technique in the development of new efficient DDS as well as pointing out some possibilities to adapt the synthesis procedures to create systems compatible with both the relative instable drug molecules, especially of peptide nature, and the sensitive physiological tissues with which MIP-based DDS would enter into contact when administered. The prospects for future development are also analysed.     

Modified linear polyethylenimine-cholesterol conjugates for DNA complexation

Linear polyethylenimine (LPEI) is an effective nonviral gene carrier with transfection levels equal or above branched polyethylenimine (BPEI) and exhibits a lower cytotoxicity profile than BPEI. High molecular weight LPEI M(w) 25 k was modified with cholesterol in three different geometries: linear shaped (L), T-shaped (T), and a combined linear/T-shaped (LT) forming the LPEI-cholesterol (LPC) conjugates LPC-L, LPC-T, and LPC-LT, respectively. Physical characterization of LPC/pDNA complexes included particle size, zeta potential, DNase protection, mIL-12 p70 expression, and cytotoxicity. The particle size was further confirmed by atomic force microscopy (AFM). The LPC-T/pDNA complexes were optimal at N/P 10/1 that resulted in a particle size of approximately 250 nm, which was confirmed by AFM, and a surface charge of +10 mV. These complexes also effectively protected the pDNA for up to 180 min in the presence of DNase I. B16-F0 cells transfected with LPC-L and LPC-T showed protein expression levels higher than LPEI alone and twice that of BPEI but without any significant loss in cell viability. These results were confirmed with EGFP flow cytometry and transfection of Renca cells. The differences in rates of transfection of the LPC/pDNA complexes is due in part to conformational changes from the point of complex formation to interaction with the plasma membrane. These conformation changes provide protection for unprotonated secondary amines in the LPEI backbone by hydrophobic protection of the cholesterol moiety that we termed "unprotonated reserves". Finally, we show that LPC conjugates exploit receptor-mediated endocytosis via the LDL-R pathway with transgene expression levels decreasing nearly 20% after saturating the LDL-R sites on MCF-7 cells with hLDL-R-Ab.     

Wheat germ agglutinin functionalized complexation hydrogels for oral insulin delivery

Insulin was loaded into hydrogel microparticles after two hours with loading efficiencies greater than 70% for both poly(methacrylic acid-grafted-ethylene glycol) (P(MAA-g-EG)) and poly(methacrylic acid-grafted-ethylene glycol) functionalized with wheat germ agglutinin (P(MAA-g-EG) WGA). The pH-responsive release results demonstrated that the pH shift from the stomach to the small intestine can be used as a physiologic trigger to release insulin from P(MAA-g-EG) and P(MAA-g-EG) WGA microparticles, thus limiting release of insulin into the acidic environment of the stomach. Microplates were successfully treated with PGM to create a surface that allowed for specific binding between mucins and lectins. The 1% PGM treatment followed by a 2 h BSA blocking step gave the most consistent results when incubated with F-WGA. In addition, the PGM-treated microplates were shown to create specific interactions between F-WGA and the PGM by use of a competitive carbohydrate. The 1% PGM treated microplates were also used to show that adhesion was improved in the P(MAA-g-EG) WGA microparticles over the P(MAA-g-EG) microparticles. The interaction between the PGM-treated microplate and P(MAA-g-EG) WGA was again shown to be specific by adding a competitive carbohydrate, while the interaction between P(MAA-g-EG) and the PGM-treated microplate was nonspecific. Cellular monolayers were used as another method for demonstrating that the functionalized microparticles increase adhesion over the nonfunctionalized microparticles. This work has focused on improving the mucoadhesive nature of P(MAA-g-EG) by functionalizing these hydrogel carriers with wheat germ agglutinin (WGA) to create a specific mucosal interaction and then evaluating the potential of these carriers as oral insulin delivery systems by in vitro methods. From these studies, it is concluded that the addition of the WGA on the microparticles produces a specific adhesion to carbohydrate-containing surfaces and that P(MAA-g-EG) WGA shows great promise as an oral insulin delivery system.     

Photocrosslinkable starch-based polymers for ophthalmologic drug delivery

This study focused on the development and characterization of a starch-based polymer with urethane linkages to be used as a controlled drug delivery system for biomedical applications. Starch was modified with 2-isocyanatoethyl methacrylate in order to obtain a polymer containing carbon-carbon double bonds in its structure. This modified starch was then used to produce films by UV irradiation using Irgacure 2959 (CIBA) as the photoinitiator. The modified polymer was characterized by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. The swelling capacity, in artificial lachrymal fluid (performed both at room temperature and physiological temperature), and water contact angles measurements were determined. The in vitro biodegradation in artificial lachrymal fluid supplemented with lysozyme was also studied. Scanning electronic microscopy (SEM) was used to characterize the morphology of the materials immediately after synthesis and after biodegradation. Timolol maleate and sodium flurbiprofen were immobilized by adsorption and their in vitro release profiles were followed spectroscopically.     

Advanced polymers for DNA separation

Recent research to improve matrices for DNA separation has resulted in the development of advanced polymers for use in capillary electrophoresis and, more generally, for electrophoresis in microchannels. To date, the most commonly used matrix is linear polyacrylamide (LPA). Unfortunately, the high-molecular weight LPA solutions required for achieving good resolution lead to very viscous solutions. Moreover, the coating ability of LPA is very poor. For these reasons, many research groups have developed low-viscosity matrices, which make microchannel filling easier, and self-coating matrices, which are able to reduce efficiently the electro-osmotic flow and the interaction of DNA with the capillary wall. To this purpose, thermo-adjustable viscosity polymers represent a very clever and interesting class of matrices.     

Engineering biodegradable and multifunctional peptide-based polymers for gene delivery

The complex nature of in vivo gene transfer establishes the need for multifunctional delivery vectors capable of meeting these challenges. An additional consideration for clinical translation of synthetic delivery formulations is reproducibility and scale-up of materials. In this review, we summarize our work over the last five years in developing a modular approach for synthesizing peptide-based polymers. In these materials, bioactive peptides that address various barriers to gene delivery are copolymerized with a hydrophilic backbone of N-(2-hydroxypropyl)methacrylamide (HPMA) using reversible-addition fragmentation chain-transfer (RAFT) polymerization. We demonstrate that this synthetic approach results in well-defined, narrowly-disperse polymers with controllable composition and molecular weight. To date, we have investigated the effectiveness of various bioactive peptides for DNA condensation, endosomal escape, cell targeting, and degradability on gene transfer, as well as the impact of multivalency and polymer architecture on peptide bioactivity.     

Polymorphism of pyridinium amphiphiles for gene delivery: influence of ionic strength, helper lipid content, and plasmid DNA complexation

Two double-tailed pyridinium cationic amphiphiles, differing only in the degree of unsaturation of the alkyl chains, have been selected for a detailed study of their aggregation behavior, under conditions employed for transfection experiments. The transfection efficiencies of the two molecules are remarkably different, especially when combined with 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) as helper lipid. The phase behavior of the cationic amphiphile/DOPE mixtures have been studied using (31)P- and (2)H-NMR (on deuterated cationic amphiphiles) as main techniques, to monitor independently the behavior of the two components. In water, the lamellar organization is dominant for both the surfactants in their mixtures with the helper lipid. In HEPES saline buffer (HBS), the mixtures of the unsaturated surfactant form inverted phases and, in particular, stable H(II) phases for DOPE contents > or =30 mol %. By contrast, the saturated surfactant does not form homogeneously mixed inverted phases in mixtures with DOPE at room temperature. However, mixed inverted phases are observed for this system at higher temperatures and, after mixing has been achieved by heating, the metastable mixed phases remain present for several hours at 5 degrees C. At 35 degrees C the dominant phase is the cubic phase. The lipoplex composed of equimolar mixtures of the unsaturated surfactant with DOPE and plasmid DNA was found to be organized in highly curved bilayers.     

Microencapsulation of nanoparticulate complexes of DNA with cationic lipids and polymers in pectin beads for targeted gene delivery

Nanoparticulate complexes of plasmid DNA (pDNA) with cationic liposomes/polymer, of approx 200 nm diameter, were encapsulated with a high degree of efficiency within calcium pectinate gel beads. Electron microscopy showed the DNA nanocomplexes to be evenly distributed throughout the gel matrix. Controlled release of pDNA-lipid nanocomplexes was achieved by the action of pectinase enzymes, whereas release of naked and polymer-complexed DNA was found to be more greatly influenced by the swelling behavior of the polysaccharide matrices in buffer alone. Physical degradation of pDNA within pectin beads was found to be accelerated during bead drying, most probably as a result of shear forces generated within the gel matrices by the evaporation of water. Plasmid complexation with cationic liposomes provided a greater degree of protection for the DNA during bead drying than complexation with cationic polymer, and was shown to successfully transfect cultured cells after release from the beads, via the action of pectinase. Observations concerning the physical stability of nanocomplexed pDNA, and its encapsulation within and release from pectin gel beads, are discussed with reference to the electrostatic interactions existing between the various components.     

Functionalized amphiphilic hyperbranched polymers for targeted drug delivery

Amphiphilic hyperbranched core-shell polymers with folate moieties as the targeting groups were synthesized and characterized. The core of the amphiphilic polymers was hyperbranched aliphatic polyester Boltorn H40. The inner part and the outer shell of the amphiphilic polymers were composed of hydrophobic poly(epsilon-caprolactone) segments and hydrophilic poly(ethylene glycol) (PEG) segments, respectively. To achieve tumor cell targeting property, folic acid was further incorporated to the surface of the amphiphilic polymers via a coupling reaction between the hydroxyl group of the PEG segment and the carboxyl group of folic acid. The polymers were characterized by (1)H NMR, (13)C NMR, and combined size-exclusion chromatography and multiangle laser light scattering analysis. The nanoparticles of the amphiphilic polymers prepared by dialysis method were characterized by transmission electron microscopy and particle size analysis. Two antineoplastic drugs, 5-fluorouracil and paclitaxel, were encapsulated into the nanoparticles. The drug release property and the targeting of the drug-loaded nanoparticles to different cells were evaluated in vitro. The results showed the drug-loaded nanoparticles exhibited enhanced cell inhibition because folate targeting increased the cytotoxicity of drug-loaded nanoparticles against folate receptor expressing tumor cells.     

Hydrophobic and electrostatic interactions between cell penetrating peptides and plasmid DNA are important for stable non‐covalent complexation and intracellular delivery

Cell penetrating peptides are useful tools for intracellular delivery of nucleic acids. Delivery of plasmid DNA, a large nucleic acid, poses a challenge for peptide mediated transport. The paper investigates and compares efficacy of five novel peptide designs for complexation of plasmid DNA and subsequent delivery into cells. The peptides were designed to contain reported DNA condensing agents and basic cell penetrating sequences, octa‐arginine (R₈) and CHK₆HC coupled to cell penetration accelerating peptides such as Bax inhibitory mutant peptide (KLPVM) and a peptide derived from the Kaposi fibroblast growth factor (kFGF) membrane translocating sequence. A tryptophan rich peptide, an analogue of Pep‐3, flanked with CH₃ on either ends was also a part of the study. The peptides were analysed for plasmid DNA complexation, protection of peptide–plasmid DNA complexes against DNase I, serum components and competitive ligands by simple agarose gel electrophoresis techniques. Hemolysis of rat red blood corpuscles (RBCs) in the presence of the peptides was used as a measure of peptide cytotoxicity. Plasmid DNA delivery through the designed peptides was evaluated in two cell lines, human cervical cancer cell line (HeLa) and (NIH/3 T3) mouse embryonic fibroblasts via expression of the secreted alkaline phosphatase (SEAP) reporter gene. The importance of hydrophobic sequences in addition to cationic sequences in peptides for non‐covalent plasmid DNA complexation and delivery has been illustrated. An alternative to the employment of fatty acid moieties for enhanced gene transfer has been proposed. Comparison of peptides for plasmid DNA complexation and delivery of peptide–plasmid DNA complexes to cells estimated by expression of a reporter gene, SEAP. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Dendritic star polymers for efficient DNA binding and stimulus-dependent DNA release

Water-soluble core-shell star polymers consisting of a dendritic polyphenylene core and an outer shell containing a defined number of amino groups have been synthesized via atom transfer radical polymerization (ATRP). All macromolecules efficiently interacted with a diverse set of DNA fragments, and stable complexes were formed and visualized by atomic force microscopy. The observed tight binding of DNA, which was found in the sub-nanomolar range, was mainly attributed to strong electrostatic interactions. Complex stoichiometries between the polyelectrolytes were controlled via the number of amino groups of the star polymers, and well-defined nanoscopic architectures were formed. DNA was released from the complexes after treatment with high concentrations of sodium chloride in aqueous solution. Such star polymers, which allow the binding and release of DNA, represent attractive candidates for the development of novel anion-exchange resins for DNA purification or as nonviral vector systems for gene delivery.     

Linear cationic click polymers/DNA nanoparticles: in vitro structure-activity relationship and in vivo evaluation for gene delivery

The aim of this work was to explore the structure--activity relationships (SAR) of a series of novel linear cationic click polymers with various structures for in vitro gene delivery and in vivo gene transfer. The experimental results revealed that the minimal structure variation could result in a crucial effect on DNA-binding ability, buffering capacity, and the cellular delivery capacity of polymer, all of which brought about the obvious effects on their transfection efficiencies. The polymer synthesized from diazide monomer containing bis-ethylenediamine unit and dialykene monomer containing bis-ethylene glycol unit (B(2)) could effectively condense DNA into complex nanoparticles (B(2)Ns), which showed the highest in vitro transfection efficiency. The biodistribution and transfection efficiency of B(2)Ns in nude mice bearing tumor demonstrated the ability of effectively delivering DNA into tumor tissue. These results implied that this gene vector based on linear cationic click polymer could be a promising gene delivery system for tumor gene therapy.     

Microbial polymers as a degradable carrier for pesticide delivery

The possibility of use of polyhydroxyalkanoates (PHAs), biodegradable microbial polyesters, as a carrier for pesticides (α-hexachlorcyclohexane and lindane) for targeted and controlled delivery of these compounds to soil was investigated. The kinetics of polymer degradation and the dynamics of pesticide release from the extended-release formulations was studied. It is shown that pesticides embedded in a degradable polymer (PHA) carrier are released gradually and slowly, without surges, as the polymer is degraded by the soil micro-flora. The microbial soil component actively responded to the addition of the polymer as an additional nutrient substrate: the latter was degraded and then utilized. The rate of the pesticide release to the soil can be regulated by varying the polymer-pesticide ratio.     

Biocatalytic route to sugar-PEG-based polymers for drug delivery applications

Sugar-PEG-based polymers were synthesized by enzymatic copolymerization of 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-benzylidene-β-L-threo-pentofuranose/4-C-hydroxymethyl-1,2-O-isopropylidene-3-O-pentyl-β-L-threo-pentofuranose with PEG-600 dimethyl ester using Novozyme-435 (Candida antarctica lipase immobilized on polyacrylate). Carbohydrate monomers were obtained by the multistep synthesis starting from diacetone-D-glucose and PEG-600 dimethyl ester, which was in turn obtained by the esterification of the commercially available PEG-600 diacid. Aggregation studies on the copolymers revealed that in aqueous solution those polymers bearing the hydrophobic pentyl/benzylidene moiety spontaneously self-assembled into supramolecular aggregates. The critical aggregation concentration (CAC) of polymers was determined by surface tension measurements, and the precise size of the aggregates was obtained by dynamic light scattering. The polymeric aggregates were further explored for their drug encapsulation properties in buffered aqueous solution of pH 7.4 (37 °C) using nile red as a hydrophobic model compound by means of UV/vis and fluorescence spectroscopy. There was no significant encapsulation in polymer synthesized from 4-C-hydroxymethyl-1,2-O-isopropylidene-β-L-threo-pentofuranose because this sugar monomer does not contain a big hydrophobic moiety as the pentyl or the benzylidene moiety. Nile red release study was performed at pH 5.0 and 7.4 using fluorescence spectroscopy. The release of nile red from the polymer bearing benzylidene moiety and pentyl moiety was observed with a half life of 3.4 and 2.0 h, respectively at pH 5.0, whereas no release was found at pH 7.4.     

Synthesis of folate-functionalized RAFT polymers for targeted siRNA delivery

Receptor-mediated, cell-specific delivery of siRNA enables silencing of target genes in specific tissues, opening the door to powerful therapeutic options for a multitude of diseases. However, the development of delivery systems capable of targeted and effective siRNA delivery typically requires multiple steps and the use of sophisticated, orthogonal chemistries. Previously, we developed diblock copolymers consisting of dimethaminoethyl methacrylate-b-dimethylaminoethyl methacrylate-co-butyl methacrylate-co-propylacrylic acid as potent siRNA delivery systems that protect siRNA from enzymatic degradation and enable its cytosolic delivery through pH-responsive, endosomolytic behavior. (1, 2) These architectures were polymerized using a living radical polymerization method, specifically reversible addition-fragmentation chain transfer (RAFT) polymerization, which employs a chain transfer agent (CTA) to modulate the rate of reaction, resulting in polymers with low polydispersity and telechelic chain ends reflecting the chemistry of the CTA. Here we describe the straightforward, facile synthesis of a folate receptor-targeted diblock copolymer siRNA delivery system because the folate receptor is an attractive target for tumor-selective therapies as a result of its overexpression in a number of cancers. Specifically, we detail the de novo synthesis of a folate-functionalized CTA, use the folate-CTA for controlled polymerizations of diblock copolymers, and demonstrate efficient, specific cellular folate receptor interaction and in vitro gene knockdown using the folate-functionalized polymer.     

Novel functionalized biodegradable polymers for nanoparticle drug delivery systems

We have prepared and screened a library of novel functionalized polymers for development of nanoparticle drug delivery systems. The polymer backbone consisting of two ester-linked, nontoxic, biological monomers, glycerol and adipic acid, was prepared using a hydrolytic enzyme. The specificity of the chosen enzyme yields a linear polymer with one free pendant hydroxyl group per repeat unit, which can be further functionalized. This protocol gives control over the backbone polymer molecular weight, together with the ability to incorporate various amounts of different fatty acyl substituents. These functionalized polymers are able to self-assemble into well-defined small particles of high homogeneity with a very low toxicity. They are able to incorporate a water soluble drug, dexamethasone phosphate, with a high efficiency and drug loading which varies with the polymer specification. The above characteristics strongly suggest that these polymers could be developed into useful nanoparticulate drug delivery systems.     

Influence of hydrophilic polymers on celecoxib complexation with hydroxypropyl beta-cyclodextrin

Complexation of celecoxib with hydroxypropyl beta-cyclodextrin (HPbetaCD) in the presence and absence of 3 hydrophilic polymers-polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG)-was investigated with an objective of evaluating the effect of hydrophilic polymers on the complexation and solubilizing efficiencies of HPbetaCD and on the dissolution rate of celecoxib from the HPbetaCD complexes. The phase solubility studies indicated the formation of celecoxib-HPbetaCD inclusion complexes at a 1:1M ratio in solution in both the presence and the absence of hydrophilic polymers. The complexes formed were quite stable. Addition of hydrophilic polymers markedly enhanced the complexation and solubilizing efficiencies of HPbetaCD. Solid inclusion complexes of celecoxib-HPbetaCD were prepared in 1:1 and 1:2 ratios by the kneading method, with and without the addition of hydrophilic polymers. The solubility and dissolution rate of celecoxib were significantly improved by complexation with HPbetaCD. The celecoxib-HPbetaCD (1:2) inclusion complex yielded a 36.57-fold increase in the dissolution rate of celecoxib. The addition of hydrophilic polymers also markedly enhanced the dissolution rate of celecoxib from HPbetaCD complexes: a 72.60-, 61.25-, and 39.15-fold increase was observed with PVP, HPMC, and PEG, respectively. Differential scanning calorimetry and X-ray diffractometry indicated stronger drug amorphization and entrapment in HPbetaCD because of the combined action of HPbetaCD and the hydrophilic polymers.     

Biocompatible controlled release polymers for delivery of polypeptides and growth factors

The development of biocompatible, controlled release systems for macromolecules has provided the opportunity for researchers and clinicians to target and deliver, on site, biologically active factors. This advance has also facilitated the purification and characterization of a number of important biomolecules. These systems include controlled release delivery systems which release proteins through porous polymer matrices, degradable polymeric delivery systems, and modulated polymer release systems. These areas of research will be reviewed with regards to their design, release kinetics, and biocompatibilities. The utilization of these systems to release such biologically important polypeptides as growth factors (e.g., fibroblast growth factor, epidermal growth factor, transforming growth factor-B) as well as a number of important inhibitory factors (e.g., nitrosoureas, angiogenesis inhibitors) in both in vivo and in vitro studies will be discussed.