Protein conjugation with amphiphilic block copolymers for enhanced cellular delivery

Modification of a model protein, horseradish peroxidase (HRP), with amphiphilic block copolymer poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide) (Pluronic), was previously shown to enhance the transport of this protein across the blood-brain barrier in vivo and brain microvessel endothelial cells in vitro. This work develops procedures for synthesis and characterization of HRP with Pluronic copolymers, having different lengths of poly(ethylene oxide) (PEO) and poly(propylene oxide) (PPO) blocks. Four monoamine Pluronic derivatives (L81, P85, L121, P123) were synthesized and successfully conjugated to a model protein, HRP, via biodegradable or nondegradable linkers (dithiobis(succinimidyl propionate) (DSP), dimethyl 3,3'-dithiobispropionimidate (DTBP), and disuccinimidyl propionate (DSS)). The conjugation was confirmed by HRP amino group titration, matrix-assisted laser desorption/ionization-time of flight spectroscopy, and cation-exchange chromatography. HRP conjugates containing an average of one to two Pluronic moieties and retaining in most cases over 70% of the activity were synthesized. Increased cellular uptake of these conjugates was demonstrated using the Mardin-Derby canine kidney cell line and primary bovine brain microvessel endothelial cells. The optimal modifications included Pluronic L81 and P85. These copolymers have shorter PPO chains compared to Pluronic P123 and L121, which were less efficient. There was little if any dependence of the uptake on the length of the hydrophilic PEO block for the optimal modifications. The proposed modifications may be used to increase cellular uptake of other proteins.     

Enhanced bone marrow stromal cell adhesion and growth on segmented poly(ether ester)s based on poly(ethylene oxide) and poly(butylene terephthalate)

In previous studies in rats and goats, hydrophilic compositions of the PEOT/PBT block copolymer family have shown in vivo calcification and bone bonding. These copolymers are therefore interesting candidates as scaffolding materials in bone tissue engineering applications. Model studies using goat bone marrow stromal cells, however, showed that it was not possible to culture bone marrow stromal cells in vitro on these hydrophilic copolymers. In this paper two ways of surface modifying these materials to improve in vitro bone marrow stromal cell attachment and growth are discussed. Two different approaches are described: (1) blending of hydroxyapatite (HA) followed by CO(2) gas plasma etching; (2) surface modification using CO(2) gas plasma treatments. It was observed that not only HA but also the CO(2) plasma treatment by itself has a positive effect on bone marrow stromal cell attachment and growth. Gas plasma treatment appeared to be the most successful approach, resulting in a large increase in the amount of bone marrow stromal cells present on the surface (determined by a DNA assay). The amount of DNA present on the plasma-treated copolymer 1000/70/30 PEOT/PBT, based on poly(ethylene oxide, M(w) = 1000, 70 m% soft segment), was comparable to the amount present on PDLLA and significantly higher than the amount present on PCL after 7 days of cell culturing. The fact that after gas plasma treatment bone marrow stromal cells do attach to PEOT/PBT copolymers, enables in vitro bone marrow stromal cell culturing, making bone tissue engineering applications of these materials possible.     

Synthesis and characterization of poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymers bearing a mannose group at the PEO chain end

A poly(ethylene oxide)-block-poly(methylidene malonate 2.1.2) block copolymer (PEO-b-PMM 2.1.2) bearing a mannose moiety at the poly(ethylene oxide) chain end was synthesized by sequential anionic polymerization of ethylene oxide (EO) and methylidene malonate 2.1.2 (MM 2.1.2), followed by a coupling reaction between its poly(ethylene oxide) amino- or aldehyde-end group and a sugar derivative. Different coupling procedures, either in organic media or in aqueous micellar solutions, were examined in order to optimize the poly(ethylene oxide) end-glycosylation yield. The micellar size of the functionalized block copolymers was determined by dynamic light scattering.     

Temperature-controlled properties of DNA complexes with poly(ethylenimine)-graft-poly(N-isopropylacrylamide)

End-functionalized poly(N-isopropylacrylamide) (PNIPA) was synthesized by living free radical polymerization and conventional free radical polymerization and was used to prepare graft copolymers with poly(ethylenimine) (PEI). The copolymers exhibited lower critical solution temperature (LCST) behavior between 30 and 32 degrees C and formed complexes with plasmid DNA. The LCST of the copolymers in the DNA complexes increased slightly to approximately 34-35 degrees C. Cytotoxicity of the copolymers was evaluated by measuring lactate dehydrogenase (LDH) release from cells. The copolymers exhibited temperature-dependent toxicity, with higher levels of LDH release observed at temperatures above the LCST. Cellular uptake and transfection activity of the DNA complexes with the PEI-g-PNIPA copolymers were lower than those of the control PEI/DNA complexes at temperature below the LCST but increased to the PEI/DNA levels at temperatures above the LCST.     

Separation and recovery of DNA fragments by electrophoresis through a thermoreversible hydrogel composed of poly(ethylene oxide) and poly(propylene oxide)

We have synthesized and characterized a thermoreversible hydrogel of multiplied block copolymers, composed of poly(ethylene oxide) and poly(propylene oxide), for DNA electrophoresis. The aqueous solution of block copolymers turned into a hydrogel upon heating at temperatures above 10-11 degrees C, whereas it reverted into a solution upon cooling. Linear double-stranded DNA molecules migrated through the gel matrices at a rate that was inversely proportional to the logarithm of the DNA length. The hydrogel is most effective for separating DNA fragments in the 10- to 2000-bp range. The resolving range lay in-between the effective ranges of polyacrylamide and agarose gel electrophoreses of DNA. The gel slices containing DNA fragments were liquefied by cooling on ice, and the DNA was precipitated with ethanol. No contaminants that inhibit enzymatic reactions were found in the DNA recovered from the hydrogel. Plasmid DNA recovered from the hydrogel was recircularized with T4 DNA ligase and yielded highly efficient Escherichia coli transformation. Therefore, thermoreversible gel electrophoresis will be a useful method for DNA separation and isolation in recombinant DNA technology.     

Amphiphilic block copolymers enhance cellular uptake and nuclear entry of polyplex-delivered DNA

This work for the first time demonstrates that synthetic polymers enhance uptake and nuclear import of plasmid DNA (pDNA) through the activation of cellular trafficking machinery. Nonionic block copolymers of poly(ethylene oxide) and poly(propylene oxide), Pluronics, are widely used as excipients in pharmaceutics. We previously demonstrated that Pluronics increase the phosphorylation of IkappaB and subsequent NFkappaB nuclear localization as well as upregulate numerous NFkappaB-related genes. In this study, we show that Pluronics enhance gene transfer by pDNA/polycation complexes ("polyplexes") in a promoter-dependent fashion. Addition of Pluronic P123 or P85 to polyethyleneimine-based polyplexes had little effect on polyplex particle size but significantly enhanced pDNA cellular uptake, nuclear translocation, and gene expression in several cell lines. When added to polyplex-transfected cells after transfection, Pluronics enhanced nuclear import of pDNA containing NFkappaB binding sites, but have no effect on import of pDNA without these sites. Altogether, our studies suggest that Pluronics rapidly activate NFkappaB, which binds cytosolic pDNA that possesses promoters containing NFkappaB binding sites and consequently increase nuclear import of pDNA through NFkappaB nuclear translocation.     

Coating of DNA/poly(L-lysine) complexes by covalent attachment of poly[N-(2-hydroxypropyl)methacrylamide

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers (pHPMA) containing 4-nitrophenyl ester (ONp) or thiazolidine-2-thione (TT) reactive groups in side chains and telechelic/semitelechelic pHPMA with TT groups were designed as highly hydrophilic biocompatible polymers suitable for chemical coating of polyelectrolyte-based DNA-containing nanoparticles bearing amino groups on the surface. The course of the coating reaction carried out in aqueous solution was evaluated on model self-assembling polyelectrolyte DNA/poly(L-lysine) (DNA/PLL) complexes either by monitoring the amount of residual polymer reactive groups by UV spectroscopy or by monitoring changes in the weight-average molecular weight and hydrodynamic size of the complexes using light scattering methods. Physicochemical stability of the coated complexes in buffered saline solution was also investigated. Contrary to uncoated particles, the coated complexes showed remarkable stability to aggregate in 0.15 M NaCl. Coating with pHPMA had practically no effect on the size distribution of the most stable complexes prepared by complexation of DNA with high-molecular-weight PLL (M(w) = 134 000) as shown by dynamic light scattering. The coating reaction was faster and more efficient with multivalent HPMA copolymers containing TT reactive groups than that with HPMA copolymers containing ONp groups.     

Thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive degradable polymer

We report a reverse thermogelling poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer as a thiol-sensitive biodegradable polymer. The poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) aqueous solutions studied in this research underwent sol-gel-sol or sol-gel-sol-gel transition depending on the molecular weight and concentration of the polymer, whereas the corresponding disulfide multiblock copolymer aqueous solutions underwent sol-gel transition as the temperature increased in a range of 0-60 degrees C. The hydrophobic dye solubilization and dynamic light scattering of the polymer aqueous solution suggest that the poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)s undergo unimer (3 nm) to micelle (12 nm) transition, whereas the disulfide multiblock copolymers undergo unimer (6 nm) to aggregated polymer (600 nm) transition as the temperature increases. The gel duration increased from 6 h (poly(ethylene oxide-b-propylene oxide-b-ethylene oxide)) to more than 12 days (the corresponding disulfide multiblock copolymer) in phosphate buffer saline, and the gel duration of the latter depended on the glutathione concentration of the medium. The model drug, paclitaxel, was released from the in-situ-formed poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) disulfide multiblock copolymer gel in a glutathione concentration-sensitive manner.     

Mannosylated poly(ethylene oxide)-b-poly(epsilon-caprolactone) diblock copolymers: synthesis, characterization, and interaction with a bacterial lectin

A novel bioeliminable amphiphilic poly(ethylene oxide)-b-poly(epsilon-caprolactone) (PEO-b-PCL) diblock copolymer end-capped by a mannose residue was synthesized by sequential controlled polymerization of ethylene oxide and epsilon-caprolactone, followed by the coupling of a reactive mannose derivative to the PEO chain end. The anionic polymerization of ethylene oxide was first initiated by potassium 2-dimethylaminoethanolate. The ring-opening polymerization of epsilon-caprolactone was then initiated by the omega-hydroxy end-group of PEO previously converted into an Al alkoxide. Finally, the saccharidic end-group was attached by quaternization of the tertiary amine alpha-end-group of the PEO-b-PCL with a brominated mannose derivative. The copolymer was fully characterized in terms of chemical composition and purity by high-resolution NMR spectroscopy and size exclusion chromatography. Furthermore, measurements with a pendant drop tensiometer showed that both the mannosylated copolymer and the non-mannosylated counterpart significantly decreased the dichloromethane/water interfacial tension. Moreover, these amphiphilic copolymers formed monodisperse spherical micelles in water with an average diameter of approximately 11 nm as measured by dynamic light scattering and cryo-transmission electron microscopy. The availability of mannose as a specific recognition site at the surface of the micelles was proved by isothermal titration microcalorimetry (ITC), using the BclA lectin (from Burkholderia cenocepacia), which interacts selectively with alpha-D-mannopyranoside derivatives. The thermodynamic parameters of the lectin/mannose interaction were extracted from the ITC data. These colloidal systems have great potential for drug targeting and vaccine delivery systems.     

Synthesis and characterization of six-arm star poly(delta-valerolactone)-block-methoxy poly(ethylene glycol) copolymers

Novel amphiphilic six-arm star diblock copolymers based on biocompatible and biodegradable poly(delta-valerolactone) (PVL) and methoxy poly(ethylene glycol) (MePEG) were synthesized by a two-step process. First, the hydrophobic star-shaped PVL with hydroxyl terminated functional groups was synthesized using a multifunctional alcohol, dipentaerythritol (DPE), as the initiator and fumaric acid as the catalyst. The amphiphilic six-arm star copolymer of poly(delta-valerolactone)-b-methoxy poly(ethylene glycol), (PVL-b-MePEG)(6), was then synthesized by coupling the hydroxyl terminated six-arm PVL homopolymer with alpha-methoxy-omega-chloroformate-poly(ethylene glycol) (MePEG-COCl). (1)H NMR and GPC analyses confirmed the successful synthesis of star-shaped copolymers with predicted compositions and narrow molecular weight distributions. DSC analysis revealed that the glass transition temperatures of the star PVL homopolymers with M(n) between 5000 and 49 000 are not dependent on their molecular weights, whereas the melting temperatures of both the PVL homopolymers and the amphiphilic (PVL-b-MePEG)(6) copolymers increase with an increase in the PVL molecular weight. Micelles were prepared from the (PVL-b-MePEG)(6) copolymers via the dialysis method and found to have effective mean diameters ranging from 10 to 45 nm, depending on the copolymer composition. In addition, the (PVL-b-MePEG)(6) copolymers having lower PVL content were found to form micelles with a narrow monomodal size distribution, whereas the copolymers having higher PVL content tended to form aggregates with a bimodal size distribution. The noncytotoxicity of the copolymers was also confirmed in CHO-K1 fibroblast cells using a cell viability assay, indicating that the (PVL-b-MePEG)(6) copolymers are suitable for biomedical applications such as drug delivery.     

Hydroxypropyl cellulose/poly(ethylene glycol)-co-poly(propylene glycol) aqueous two-phase systems: system characterization and partition of cells and proteins.

Novel aqueous polymeric two-phase systems are described. These systems are formed by mixing hydroxypropyl cellulose (molecular mass 100,000, trade name Klucel L) with poly(ethylene glycol)-co-poly(propylene glycol) copolymer [molecular mass 6,500, poly(propylene glycol) content 50% w/w, trade name Pluronic P105], in a saline buffer. The phase diagram was measured and the interfacial tensions, phase separation times, and lower phase viscosities of three phase systems having constant Pluronic P105 concentration but varying in Klucel L concentration were determined. The partition behavior of a representative cell, bacterium, and protein and the affinity ligand-mediated alteration in the partition behavior of a protein from a yeast extract protein mixture were also characterized. The results suggest that Klucel L/Pluronic P105 phase systems may be cost-effective substitutes for, or complements to, existing aqueous polymeric phase systems. The physical characterization and representative partition data reported here should facilitate application of these new systems.     

Synthesis and characterization of self-assembling block copolymers containing bioadhesive end groups

3,4-Dihydroxyphenyl-L-alanine (DOPA) is an unusual amino acid found in mussel adhesive proteins (MAPs) that is believed to lend adhesive characteristics to these proteins. In this paper, we describe a route for the conjugation of DOPA moieties to poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) block copolymers. Hydroxyl end groups of PEO-PPO-PEO block copolymers were activated by N,N'-disuccinimidyl carbonate and then reacted with DOPA or its methyl ester with high coupling efficiencies from both aqueous and organic solvents. DOPA-modified PEO-PPO-PEO block copolymers were freely soluble in cold water, and dye partitioning and differential scanning calorimetry analysis of these solutions revealed that the copolymers aggregated into micelles at a characteristic temperature that was dependent on block copolymer composition and concentration in solution. Oscillatory rheometry demonstrated that above a block copolymer concentration of approximately 20 wt %, solutions of DOPA-modified PEO-PPO-PEO block copolymers exhibited sol-gel transitions upon heating. The gelation temperature could be tailored between approximately 23 and 46 degrees C by changing the composition, concentration, and molecular weight of the block copolymer. Rheological measurement of the bioadhesive interaction between DOPA-modified Pluronic and bovine submaxillary mucin indicated that DOPA-modified Pluronic was significantly more bioadhesive than unmodified Pluronic.     

Synthesis and self-association behavior of biodegradable amphiphilic poly[bis(ethyl glycinat-N-yl)phosphazene]- poly(ethylene oxide) block copolymers

Amphiphilic diblock copolymers with varying compositions of hydrophilic poly(ethylene oxide) (PEO) and hydrophobic poly[bis(ethyl glycinat-N-yl)phosphazene] (PNgly) were synthesized via the controlled cationic-induced polymerization of a phosphoranimine (Cl(3)P=NSiMe(3)) at ambient temperature using a PEO-phosphoranimine macroinitiator. The aqueous-phase transition behavior of PEO-PNgly-3 (M(n) = 10,000) and micelle formation of both PEO-PNgly-3 and PEO-PNgly-4 (M(n) = 8,500) were investigated using fluorescence techniques and dynamic light scattering. The critical micelle concentrations (cmc's) of PEO-PNgly-3 and PEO-PNgly-4 were determined to be 3 and 12 mg/L with the mean diameters of micelles being 120 and 130 nm, respectively. The hydrolytic degradation of these diblock copolymers was also studied in solution. These studies coupled with the biodegradability of the poly[bis(ethyl glycinat-N-yl)phosphazene] block to give benign products make PEO-PNgly copolymers well-suited for a wide variety of biomedical applications including novel biodegradable drug-delivery systems.     

Ionophoric activity of pluronic block copolymers

Pluronic block copolymers (triblock copolymers of poly(ethylene oxide) and poly(propylene oxide)) exhibit a chemosensitizing effect on multidrug resistant cell lines. Changes in membrane permeability are hypothesized to be responsible because inhibition of drug transport mediated by both the multidrug-resistance-associated protein and the P-glycoprotein drug efflux system has been observed. To test this hypothesis, we now have studied the ion conductivity mediated by Pluronic L61. Besides a detergent-like action, the copolymer was able to form regular channels and to exhibit carrier activity. Long living ion channels were formed by polymer oligomerization. Aggregate equilibrium was shifted toward L61 monomers and dimers, which operated as mobile carriers. Copolymer-induced membrane permeability for potassium ions (1 M KCl) was less than 10(-8) cm s(-1), whereas the permeability for uncharged doxorubicin molecules (1 mM) was equal to 5 x 10(-4) cm s(-1). The results are consistent with reports about an increased doxorubicin accumulation in cells (Venne, Li, S., Mandeville, R., Kabanov, A., and Alakhov, V. Y. (1996) Cancer Res. 56, 3626-3629). However, the increased permeability contrasts with the polymer-mediated decrease of drug efflux from cells. Preferential polymer binding to membrane proteins may mask the unspecific effect of L61 observed on lipid bilayers.     

Complexation of DNA with poly(methacryl oxyethyl trimethylammonium chloride) and Its poly(oxyethylene) grafted analogue

Intermolecular complexes of genomic polydisperse DNA with synthetic polycations have been studied. Two cationic polymers have been used, a homopolymer poly(methacryl oxyethyl trimethylammonium chloride) (PMOTAC) and its analogue grafted with poly(oxyethylene). The amount of poly(oxyethylene) grafts in the copolymer was 15 mol % and Mw of the graft was 200 g/mol. Salmon DNA (sodium salt) was used. The average molecular weight (Mw) of DNA was 10.4 x 10(6) g/mol. Conductivity, pH, and dynamic light scattering studies were used to characterize the complexes. The size and shape of the polyelectrolyte complex particles have been studied as a function of the cation-to-anion ratio in aqueous solutions of varying ionic strengths. The polyelectrolyte complexes have extremely narrow size distributions taking into account the polydispersity of the polyelectrolytes studied. The poly(oxyethylene) grafts on PMOTAC promote the formation of small colloidally stabile complex particles. Addition of salt shifts the macroscopic phase separation toward lower polycation content; that is, complexes partly phase separate with the mixing ratios far from 1:1. Further addition of salt to the turbid, partly phase separated solution results in the dissociation of complexes and the polycation and DNA dissolve as individual chains.     

Polyethylenimine-graft-poly(ethylene glycol) copolymers: influence of copolymer block structure on DNA complexation and biological activities as gene delivery system

For two series of polyethylenimine-graft-poly(ethylene glycol) (PEI-g-PEG) block copolymers, the influence of copolymer structure on DNA complexation was investigated and physicochemical properties of these complexes were compared with the results of blood compatibility, cytotoxicity, and transfection activity assays. In the first series, PEI (25 kDa) was grafted to different degrees of substitution with PEG (5 kDa) and in the second series the molecular weight (MW) of PEG was varied (550 Da to 20 kDa). Using atomic force microscopy, we found that the copolymer block structure strongly influenced the DNA complex size and morphology: PEG 5 kDa significantly reduced the diameter of the spherical complexes from 142 +/- 59 to 61 +/- 28 nm. With increasing degree of PEG grafting, complexation of DNA was impeded and complexes lost their spherical shape. Copolymers with PEG 20 kDa yielded small, compact complexes with DNA (51 +/- 23 nm) whereas copolymers with PEG 550 Da resulted in large and diffuse structures (130 +/- 60 nm). The zeta-potential of complexes was reduced with increasing degree of PEG grafting if MW >or= 5 kDa. PEG 550 Da did not shield positive charges of PEI sufficiently leading to hemolysis and erythrocyte aggregation. Cytotoxicity (lactate dehydrogenase assay) was independent of MW of PEG but affected by the degree of PEG substitution: all copolymers with more than six PEG blocks formed DNA complexes of low toxicity. Finally, transfection efficiency of the complexes was studied. The combination of large particles, low toxicity, and high positive surface charge as in the case of copolymers with many PEG 550 Da blocks proved to be most efficient for in vitro gene transfer. To conclude, the degree of PEGylation and the MW of PEG were found to strongly influence DNA condensation of PEI and therefore also affect the biological activity of the PEI-g-PEG/DNA complexes. These results provide a basis for the rational design of block copolymer gene delivery systems.     

Synthesis and characterization of poly(dimer acid-brassylic acid) copolymer and poly(dimer acid-pentadecandioic acid) copolymer

Poly(dimer acid-brassylic acid) [P(DA-BA)] copolymers and poly(dimer acid-pentadecandioic acid) [P(DA-PA)] copolymers were prepared by melt polycondensation of the corresponding mixed anhydride prepolymers. The copolymers were characterized by Fourier transform infrared (FTIR), gel permeation chromatography (GPC), differential scanning calorimetry (DSC), wide angle x-ray powder-diffraction, and thermal gravimetric analysis (TGA). In vitro studies show that all the copolymers are degradable in phosphate buffer at 37 degrees C, and leaving an oily dimer acid residue after hydrolysis for the copolymer with high content of dimer acid. The release profiles of hydrophilic model drug, ciprofloxcin hydrochloride, from the copolymers, follow first-order release kinetics. All the preliminary results suggested that the copolymer might be potentially used as drug delivery devices.     

Star-shaped poly(ethylene glycol)-block-polyethylenimine copolymers enhance DNA condensation of low molecular weight polyethylenimines

Star-shaped poly(ethylene glycol)-block-polyethylenimine [star-(PEG-b-PEI)] significantly enhance plasmid DNA condensation of low molecular weight (MW) PEIs. The star-block copolymers were prepared via a facile synthesis route using hexamethylene diisocyanate as linker between PEG and PEI blocks. NMR and FT-IR spectroscopy confirmed the structures of intermediately activated PEG and final products. Furthermore, the copolymers were characterized by size exclusion chromatography, static light scattering, and viscosimetry. Their molecular weights (M(w) 19-26 kDa) were similar to high MW PEI (25 kDa). Thermoanalytical investigations (thermogravimetric analysis, differential scanning calorimetry) were also performed and verified successful copolymer synthesis. DNA condensation with the low MW PEIs (800 and 2000 Da) and their 4- and 8-star-block copolymers was studied using atomic force microscopy, dynamic light scattering, zeta-potential measurements, and ethidium bromide (EtBr) exclusion assay. It was found that low MW PEIs formed huge aggregates (500 nm to 2 microm) in which DNA is only loosely condensed. By contrast, the star-block copolymers yielded small (80-110 nm), spherical and compact complexes that were stable against aggregation even at high ionic strength and charge neutrality. Furthermore, as revealed in the EtBr exclusion assay these star-block copolymers exhibited a DNA condensation potential as high as high MW PEI. Since these star-(PEG-block-PEI) copolymers are composed of relatively nontoxic low MW PEI and biocompatible PEG, their potential as gene delivery agents merits further investigations.     

Novel stimuli-responsive micelle self-assembled from Y-shaped P(UA-Y-NIPAAm) copolymer for drug delivery

A new amphiphilic Y-shaped copolymer, comprised of hydrophobic poly(undecylenic acid) (PUA) and hydrophilic poly(N-isopropylacrylamide) (PNIPAAm), was designed and synthesized. A cytotoxicity study revealed that P(UA-Y-NIPAAm) copolymers did not exhibit apparent inhibition impact on the proliferation of cells when the concentration of the copolymer was below 1000 mg/L. Characterization demonstrated that the P(UA-Y-NIPAAm) copolymer is thermosensitive with a lower critical solution temperature (LCST) of 31 degrees C. In water, the P(UA-Y-NIPAAm) copolymer would self-assemble into micelles with a critical micelle concentration (CMC) of 20 mg/L. Self-assembled P(UA-Y-NIPAAm) micelles exhibited a nanospherical morphology of 40 to approximately 80 nm in size. The controlled drug release behavior of the P(UA-Y-NIPAAm) micelles was further investigated, and self-assembled micelles exhibited improved properties in controlled drug release.     

Poly(organo phosphazene) nanoparticles surface modified with poly(ethylene oxide)

The use of biodegradable derivatives of poly(organo phosphazenes) for the preparation of nanoparticles and their surface modification with the novel poly(ethylene oxide) derivative of poly(organo phosphazene) has been assessed using a range of in vitro characterization methods. The nanoparticles were produced by the precipitation solvent evaporation method from the derivative co-substituted with phenylalanine and glycine ethyl ester side groups. A reduction in particle size to less than 200 nm was achieved by an increase in pH of the preparation medium. The formation (and colloidal stability) of these nanoparticles seems to be controlled by two opposite effects: attractive hydrophobic interactions between phenylalanine ester groups and electrostatic repulsions arising from the carboxyl groups formed due to (partial) hydrolysis of the ester bond(s) at the high pH of the preparation medium. The poly[(glycine ethyl ester)phosphazene] derivative containing 5000-Da poly(ethylene oxide) as 5% of the side groups was used for the surface modification of nanoparticles. Adsorbed onto the particles, the polymer produced a thick coating layer of approximately 35 nm. The coated nanoparticles exhibited reduced surface negative potential and improved colloidal stability toward electrolyte-induced flocculation, relative to the uncoated system. However, the steric stabilization provided was less effective than that of a Poloxamine 908 coating. This difference in effectiveness of the steric stabilization might indicate that, although both the stabilizing polymers possess a 5000-Da poly(ethylene oxide) moiety, there is a difference in the arrangements of these poly(ethylene oxide) chains at the particle surface. (c) 1996 John Wiley & Sons, Inc.