Novel vectors for gene delivery formed by self-assembly of DNA with poly(L-lysine) grafted with hydrophilic polymers.

Complexes formed between DNA and cationic polymers are attracting increasing attention as novel synthetic vectors for delivery of genes. We are trying to improve biological properties of such complexes by oriented self-assembly of DNA with cationic-hydrophilic block copolymers, designed to enshroud the complex within a protective hydrophilic polymer corona. Poly(L-lysine) (pLL) grafted with range of hydrophilic polymer blocks, including poly(ethylene glycol) (pEG), dextran and poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA), shows efficient binding to DNA and mediates particle self-assembly and inhibition of ethidium bromide/DNA fluorescence. The complexes formed are discrete and typically about 100 nm diameter, viewed by atomic force microscopy. Surface charges are slightly shielded by the presence of the hydrophilic polymer, and complexes generally show decreased cytotoxicity compared with simple pLL/DNA complexes. pEG-containing complexes show increased transfection activity against cells in vitro. Complexes formed with all polymer conjugates showed greater aqueous solubility than simple pLL/DNA complexes, particularly at charge neutrality. These materials appear to have the ability to regulate the physicochemical and biological properties of polycation/DNA complexes, and should find important applications in packaging of nucleic acids for specific biological applications.

Balancing polymer hydrophobicity for ligand presentation and siRNA delivery in dual function CXCR4 inhibiting polyplexes.

In the present study, a series of copolymers (PAMD-Ch) was synthesized by grafting polymeric Plerixafor/AMD3100 (PAMD) with different amounts of cholesterol and the effect of cholesterol modification on siRNA delivery was investigated. PAMD-Ch/siRNA polyplexes exhibited improved colloidal and enzymatic stability when compared with PAMD/siRNA polyplexes containing no cholesterol. PAMD-Ch with low (17 wt%) and medium (25 wt%) cholesterol content exhibited CXCR4 antagonism comparable to unmodified PAMD. Cholesterol modification increased cell uptake of siRNA polyplexes and significantly decreased sensitivity of siRNA transfection to the presence of serum. When used to deliver anticancer siRNA against polo-like kinase 1 (PLK1), polyplexes based on PAMD-Ch with 17 wt% cholesterol exhibited the highest cancer cell killing activity both in serum-free and serum-containing conditions. Overall, the results of this study validate cholesterol modified PAMD as dual-function delivery vectors suitable for efficient delivery of anticancer siRNA and simultaneous CXCR4 inhibition for combined anticancer therapies.

DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers.

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate were synthesised and used for preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study the effects of the speed of component mixing, total concentration of polymers, ionic strength of solvents, copolymer structure and content of HPMA in the copolymers on parameters of the polyelectrolyte complexes was investigated. Static and dynamic light scattering methods were used as a main tool for characterising these complexes. The presence of HPMA units in the polycation had no significant effect on its ability to form complexes with DNA, but did affect molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases whereas their molecular weight decreases with increasing content of HPMA units. The density of the complexes decreases with increasing HPMA content independently of the copolymer structure. In order to prepare stable DNA complexes containing single DNA molecule, the following rules should be observed: (1) copolymers should have a content of HPMA units higher than 40%; (2) the DNA concentrations in solutions should be kept below 4 x 10(-5) g/ml and (3) both components should be mixed together in deionized water. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. Whereas DNA complexes of all copolymers showed very good nuclease stability, the presence of a sufficiently high content of HPMA is necessary for their good stability in 0.15 M NaCl. The investigation of the stability and the interaction of DNA complexes in aqueous solutions of serum albumin and dilute human blood serum revealed adsorption of biomacromolecules on DNA complexes accompanied by significant changes in the zeta-potential which finally resulted in formation of a "protein layer" and in undesirable precipitation of DNA complexes. In in vitro transfection experiments, the transfection efficiency of DNA complexes with copolymers was always higher than that of the cationic homopolymer slightly increasing with increasing content of HPMA in the copolymers but being about 10-100-times lower than the complexes DNA-poly(L-lysine. In the cytoplasmic injections, it was observed that DNA complexes produced greater gene expression than a direct microinjection of free DNA. The block copolymer complexes were also found to be more efficient than the corresponding simple polycation complexes. In the nuclear microinjection, precisely the opposite behaviour was observed.

Stabilization of Polycation-DNA Complexes by Surface Modification with Hydrophilic Polymers.

Polycation-DNA complexes represent promising synthetic vectors for gene delivery, showing good transfection activities in vitro and safety in vivo. However, simple polycation-DNA complexes suffer from several disadvantages that limit their potential usefulness in vivo. Advances in this field thus rely on better control of the structure, colloidal, and surface properties of condensed DNA particles.

Methods for Studying the Formation of Polycation-DNA Complexes and Properties Useful for Gene Delivery.

There is an urgent requirement in the field of gene therapy for gene transfer vectors that are both safe to use and able to efficiently deliver therapeutic genes to target cells in vivo. Viral vectors, such as retrovirus, adenovirus, and herpes simplex virus, are efficient in transducing a broad range of cells, but they often lead to an inflammatory response against successfully transduced tissues, along with a strong immunogenicity of the virus itself (1). A further problem is the often expensive and laborious procedure required to produce the virus in sufficient quantities. In the past decade, several nonviral gene transfer vectors based on polycations (2) and liposomes (3,4), have been developed in order to overcome such problems. These vectors are becoming increasingly popular for use in delivering DNA to target cells both in vitro and in vivo because they are generally nonimmunogenic and easier to manufacture in bulk quantities. This chapter focuses on the use of polycations in gene delivery vectors.

Physicochemical and biological characterisation of an antisense oligonucleotide targeted against the bcl-2 mRNA complexed with cationic-hydrophilic copolymers.

The aim of this study was to evaluate the use of cationic-hydrophilic copolymers for self-assembly with antisense oligonucleotides targeted to the bcl-2 mRNA in order to improve their biocompatibility and modulation of their pharmacokinetics for greater therapeutic usefulness. Examination of the ability of poly(trimethylammonioethyl methacrylate chloride)-poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA-b-pTMAEM) block copolymers to condense the oligonucleotide by fluorescence and electrophoresis techniques showed that complexes were formed more efficiently than with copolymers containing poly(ethylene glycol) blocks grafted onto the backbone of poly(L-lysine) (pLL-g-pEG). In addition, the copolymer pTMAEM-b-pHPMA produced oligonucleotide complexes with the most favourable physicochemical properties appropriate for in vivo applications. The complexes were small (approximately 36 nm in diameter), with low surface charge as measured by zeta potential, relatively stable to physiological salt conditions and could be formed at a DNA concentration of 500 microg/ml. Complex formation with the copolymer pTMAEM-b-pHPMA or pLL-g-pEG reduced the urinary clearance of the oligonucleotide after intravenous injection into mice. However after 30 min, the oligonucleotide complexes were cleared from the bloodstream. These results indicate that for the systemic delivery of oligonucleotides the polymer-derived complexes are not stable enough for prolonged circulation. Instead, these complexes may be more suitable for localised in vivo applications.

DNA complexes with block and graft copolymers of N-(2-hydroxypropyl)methacrylamide and 2-(trimethylammonio)ethyl methacrylate.

Block and graft copolymers of N-(2-hydroxypropyl)methacrylamide (HPMA) with 2-(trimethylammonio)ethyl methacrylate (TMAEM) were synthesized for the preparation of polyelectrolyte complexes with calf thymus DNA intended for targeted delivery of genes in vivo. In this study, the effects of the poly(HPMA) content of copolymers on the parameters of the interpolyelectrolyte complexes is investigated. Static and dynamic light scattering methods were used as a main tool for characterization. The ability of the copolymers to condense DNA was studied by the ethidium bromide displacement method. The stability of the complexes against precipitation in 0.15 M NaCl and the resistance of the complexed DNA to the action of nucleases was also studied. It was found that the presence of poly(HPMA) in the copolymers has not significantly affected the ability of poly(TMAEM) parts of the copolymers to form complexes with DNA, but has an effect on molecular parameters and aggregation (precipitation) of the complexes. The size of the complexes increases with increasing poly(HPMA) content while their apparent molecular weight decreases. The complex stability against precipitation in 0.15 M NaCl strongly depends on the amount of poly(HPMA) in the copolymer structure. The presence of a sufficiently high content of poly(HPMA) is a prerequisite for achieving good stability. The structure of the complexes changes with increasing poly(HPMA) content from soft balls to the polymer coil. The density of the complexes decreases with increasing poly(HPMA) content independently of the copolymer structure. The DNA complexes of all copolymers showed very good nuclease stability.

Triggered intracellular activation of disulfide crosslinked polyelectrolyte gene delivery complexes with extended systemic circulation in vivo.

We have developed polyelectrolyte gene delivery vectors that display good extracellular stability and are activated intracellularly to permit transgene expression. The strategy comprises covalent crosslinking of primary amines in poly-L-lysine/DNA complexes with a crosslinking agent that can later be cleaved by reduction. Crosslinked complexes maintained the same size and surface charge but showed increased stability against polyelectrolyte exchange with poly-L-aspartic acid. Surface modification with polyethyleneglycol improved solubility and masked their positive surface charge. Crosslinked complexes showed 10-fold increased plasma circulation following intravenous administration to Balb/c mice. In the absence of chloroquine, the levels of transgene expression in B16F10 murine melanoma cells were similar for crosslinked and non-crosslinked complexes, however, chloroquine selectively potentiated transgene expression by the non-crosslinked complexes. Cellular uptake of the complexes was the same, irrespective of crosslinking. Following microinjection into the cytoplasm of Xenopus oocytes, or the cytoplasm or nucleus of Rat-1 fibroblasts, crosslinked complexes mediated the same transgene expression as non-crosslinked complexes, indicating crosslinked complexes are rapidly reduced and activated intracellularly. We therefore hypothesize that the lower in vitro transfection activity of crosslinked complexes in the presence of chloroquine is due to reduced transfer from endosome to cytoplasm, mainly due to increased stability against destabilization by chloroquine. The extended systemic circulation together with triggered intracellular activation makes these complexes a promising system for targeted gene delivery in vivo.

Effect of albumin and polyanion on the structure of DNA complexes with polycation containing hydrophilic nonionic block.

Self-assembling systems based on ionic complexes of DNA with block copolymer of N-(2-hydroxypropyl)methacrylamide with 2-(trimethylammonio)ethyl methacrylate were studied as systems suitable for gene delivery. In this study, the influence of albumin and polyanion on parameters of the DNA polyelectrolyte complexes in aqueous solutions was investigated. Static and dynamic light-scattering methods were used as a main tool for characterizing these interactions. It was found that albumin is not able to release free DNA, but it can rather bind to the complexes forming ternary DNA-polycation-albumin complexes with increased hydrodynamic radii of about 10 nm. Polyanion tested, sodium poly(styrenesulfonate), was able to release free DNA in the presence of a low-molecular-weight electrolyte. In the absence of a low-molecular-weight electrolyte, only formation of ternary complexes and no DNA release was observed. The in vivo biodistribution analysis of DNA complexes showed no effect of the presence of hydrophilic nonionic poly(HPMA) on the circulatory time or organ distribution. The interaction of DNA complexes with albumin and other plasma proteins was suggested to be a major reason for the short circulatory times.

Steric stabilization of poly-L-Lysine/DNA complexes by the covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide].

The concept of steric stabilization was utilized for self-assembling polyelectrolyte poly-L-lysine/DNA (pLL/DNA) complexes using covalent attachment of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] (pHPMA). We have examined the effect of coating of the complexes with pHPMA on their physicochemical stability, phagocytic uptake in vitro, and biodistribution in vivo. The coated complexes showed stability against aggregation in 0.15 M NaCl and reduced binding of albumin, chosen as a model for the study of the interactions of the complexes with plasma proteins. The presence of coating pHPMA had no effect on the morphology of the complexes as shown by transmission electron microscopy. However, results of the study of polyelectrolyte exchange reactions with heparin and pLL suggested decreased stability of the coated complexes in these types of reactions compared to uncoated pLL/DNA complexes. Coated complexes showed decreased phagocytic capture by mouse peritoneal macrophages in vitro. Decreased phagocytosis in vitro, however, did not correlate with results of in vivo study in mice showing no reduction in the liver uptake and no increase in the circulation times in the blood. We propose that the rapid plasma elimination of coated pLL/DNA complexes is a result of binding serum proteins and also of their low stability toward polyelectrolyte exchange reactions as a consequence of their equilibrium nature.

Decreased binding to proteins and cells of polymeric gene delivery vectors surface modified with a multivalent hydrophilic polymer and retargeting through attachment of transferrin.

Binding of serum proteins to polyelectrolyte gene delivery complexes is thought to be an important factor limiting bloodstream circulation and restricting access to target tissues. Protein binding can also inhibit transfection activity in vitro. In this study a multivalent reactive hydrophilic polymer has been used to inhibit protein binding. This polymer is based on poly-[N-(2-hydroxypropyl)methacrylamide] (pHPMA) bearing pendent oligopeptide (Gly-Phe-Leu-Gly) side chains terminated in reactive 4-nitrophenoxy groups (8.6 mol%). The polymer reacts with the primary amino groups of poly(L-lysine) (pLL) and produces a hydrophilic coating on the surface of pLL.DNA complexes (as measured by fluorescamine). The resulting pHPMA-coated complexes show a decreased surface charge (from +14 mV for pLL.DNA complexes to -25 mV for pHPMA-modified complexes) as measured by zeta potential analysis. The pHPMA-coated complexes also show a slightly increased average diameter (approximately 90 nm compared with 60 nm for pLL. DNA complexes) as viewed by atomic force and transmission electron microscopy and around 100 nm as viewed by photon correlation spectroscopy. They are completely resistant to protein interaction, as determined by turbidometry and SDS-polyacrylamide gel electrophoresis analysis of complexes isolated from plasma, and show significantly decreased nonspecific uptake into cells in vitro. Spare reactive ester groups can be used to conjugate targeting ligands (e.g. transferrin) on to the surface of the complex to provide a means of tissue-specific targeting and transfection. The properties of these complexes therefore make them promising candidates for targeted gene delivery, both in vitro and potentially in vivo.

Polyelectrolyte vectors for gene delivery: influence of cationic polymer on biophysical properties of complexes formed with DNA.

Cationic polymer/DNA complexes are widely used for gene delivery, although the influence of the cationic polymer on the biophysical properties of the resulting complex is poorly understood. Here, several series of cationic polymers have been used to evaluate the influence of structural parameters on properties of DNA complexes. Parameters studied included the length of side chain, charge type (primary versus tertiary and quaternary), polymer molecular weight, and charge spacing along the polymer backbone. Cationic polymers with short side chains (such as polyvinylamine) formed small complexes, resistant to destabilization by polyanions, with low surface charge, limited transfection activity, and efficient intranuclear transcription. Conversely, cationic polymers with long side chains (e.g., poly[methacryloyl-Gly-Gly-NH-(CH(2))(6)-NH(2))] showed inefficient complex formation, high positive surface charge, and better transfection activity. The effects of molecular weight varied between polymers, for example, low molecular weight poly(L-lysine) produced relatively small complexes, whereas low molecular weight poly[2-(trimethylammonio)ethyl methacrylate chloride] produced large aggregates. Polymers containing quaternary ammonium groups showed efficient complex formation but poor transfection. Finally, spreading charges widely on the polymer structure inhibited their ability to condense DNA. In summary, to achieve small, stable complexes, the use of cationic polymers with short side chains bearing primary amino groups is suggested.