New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting.

Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.

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.

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.

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.

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.

Micellization of Graft Copolymers of Alkyl Methacrylates with alpha-Methyl-omega-hydroxypoly(oxyethylene) Methacrylates.

The solution properties of graft copolymers having a poly(methyl methacrylate-co-lauryl methacrylate) backbone and poly(oxyethylene) branches of different compositions were studied by static and dynamic light scattering methods in ethanol-water mixed solvents. The graft copolymers formed multimolecular aggregates in mixed solvents with water content higher than or equal to 10 vol%. The difference in solubility of lauryl and poly(oxyethylene) side chains of copolymers seems to be a dominant factor in determining their association behavior. The micelle formation is preferred in such solvents (water content >/=30 vol%) where the difference in solubilities of LAM and PEO side chains on graft copolymers is higher than a critical one. In conditions where there is a lower difference in solubilities of the side chains (water content 10-20 vol%), the random association of copolymers dominates. Copolymers were fully soluble in ethanol with low water content (1 vol%). The highest association number of micelles and consequently the lowest polydispersity were obtained with copolymers having the longest PEO side chains (Mw = 5000 g mol-1). Copyright 1998 Academic Press.

Influence of pH on aggregation and photoproperties of n-(2-hydroxypropyl)methacrylamide copolymer-meso-chlorin e6 conjugates.

The influence of pH on the aggregation and photoproperties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing meso-chlorin e(6) monoethylenediamine (Mce(6)) attached to the copolymer via either nonbiodegradable G or biodegradable GFLG side chains was studied. Dynamic light scattering, UVIVIS and fluorescence spectroscopy, time-resolved fluorescence spectroscopy, and fluorescence quenching techniques were used. The photosensitizing efficiencies of these conjugates were also determined. The dynamic light-scattering data indicate that the intermolecular aggregation of Mce(6) species within the copolymer conjugates is not significant and is not affected by pH or loading of Mce(6) to copolymer at 5 x 10(-4) g/mL of copolymer conjugate concentration. However, intramolecular aggregation of the Mce(6) species within the copolymer conjugates does occur in aqueous buffers, as demonstrated by absorption and fluorescence measurements in ethanol-buffer mixtures. The fluorescence lifetime of excited Mce(6) was influenced by aggregation, mainly attributed to the pH and copolymer side-chain hydrophobicity. The Stern-Volmer collisional quenching constant, K(sv) iodide anion with Mce(6) species was found to be a function of pH, reflecting both the electrostatic repulsion between negatively charged Mce(6) species and iodide anions and the intramolecular aggregation of Mce(6) moieties. The extent of aggregation was found to be a function of solvent pH, loading of Mce(6) to copolymer, and side-chain hydrophobicity. The photosensitizing efficiency of the copolymer bound Mce(6), as determined through the photooxidation of furfuryl alcohol, was dominated by Mce(6) loading to copolymer and side-chain hydrophobicity, but was only slightly pH dependent. Evidently, the Mce(6) aggregation only weakly influenced the charge transfer in the process of oxygen generation.

Effect of multiple light scattering on transmitted and scattered light.

Photocount distributions have been calculated and experimentally determined for light transmitted through a dispersion of particles of varying density. From these the coherent and incoherent components of the transmitted light intensity could be determined. The coherent intensity was shown to obey the Lambert-Beer law. Autocorrelation functions of light scattered in the presence of multiple light scattering were measured and inverted by using an inverse Laplace transform technique. The single-scattering and multiple-scattering contributions can be distinguished in the corresponding spectra of decay times. The amount of multiple light scattering increases with increasing concentration of scatterers and reaches the limit of strong multiple light scattering when the transport of light through the dispersion becomes diffusive.