Thiol-yne and thiol-ene "click" chemistry as a tool for a variety of platinum drug delivery carriers, from statistical copolymers to crosslinked micelles

Statistical and block copolymers based on poly(2-hydroxyethyl methacrylate) (PHEMA) and poly[oligo(ethylene glycol) methylether methacrylate] (POEGMEMA) were modified with 4-pentenoic anhydride or 4-oxo-4-(prop-2-ynyloxy)butanoic anhydride to generate polymers with pendant vinyl or acetylene, respectively. Subsequent thiol-ene or thiol-yne reaction with thioglycolic acid or 2-mercaptosuccinic acid leads to polymers with carboxylate functionalities, which were conjugated with cisplatin (cis-diamminedichloroplatinum(II) (CDDP)) to generate a drug carrier for Pt-drugs. Only the polymers modified with 2-mercaptosuccinic acid resulted in the formation of soluble well-defined polymers with gel formation being prevented. Due to the hydrophobicity of the drug, the block copolymers took on amphiphilic character leading to micelle formation. The micelles were in addition crosslinked to further stabilize their structure. Pt-containing statistical copolymer, micelles, and crosslinked micelles were then tested regarding their cellular uptake by the A549 lung cancer cell line to show a superior uptake of crosslinked micelles. However, due to the better Pt release of the statistical copolymer, the highest cytotoxicity was observed with this type of polymer architecture.     

Amphiphilic hyperbranched fluoropolymers as nanoscopic 19F magnetic resonance imaging agent assemblies

Three hyperbranched fluoropolymers were synthesized and their micelles were constructed as potential (19)F MRI agents. A hyperbranched star-like core was first synthesized via atom transfer radical self-condensing vinyl (co)polymerization (ATR-SCVCP) of 4-chloromethyl styrene (CMS), lauryl acrylate (LA), and 1,1,1-tris(4'-(2'-bromoisobutyryloxy)phenyl)ethane (TBBPE). The polymerization gave a small core with M n of 5.5 kDa with PDI of 1.6, which served as a macroinitiator. Trifluoroethyl methacrylate (TFEMA) and tert-butyl acrylate (tBA) in different ratios were then "grafted" from the core to give three polymers with M(n) of about 120 kDa and PDI values of about 1.6-1.8. After acidolysis of the tert-butyl ester groups, amphiphilic, hyperbranched star-like polymers with M(n) of about 100 kDa were obtained. These structures were subjected to micelle formation in aqueous solution to give micelles having TEM-measured diameters ranging from 3-8 nm and DLS-measured hydrodynamic diameters from 20-30 nm. These micelles gave a narrow, single resonance by (19)F NMR spectroscopy, with a half-width of approximately 130 Hz. The T1/T2 parameters were about 500 and 50 ms, respectively, and were not significantly affected by the composition and sizes of the micelles. (19)F MRI phantom images of these fluorinated micelles were acquired, which demonstrated that these fluorinated micelles maybe useful as novel (19)F MRI agents for a variety of biomedical studies.     

Synthesis and characterization of biodegradable amphiphilic triblock copolymers containing L-glutamic acid units

A novel biodegradable amphiphilic triblock copolymer bearing pendant carboxyl groups PLGG-PEG-PLGG was successfully prepared by ring-opening copolymerization of l-lactide (LA) with (3s)-benzoxylcarbonylethyl-morpholine-2, 5-dione (BEMD) in the presence of dihydroxyl poly(ethylene glycol) (PEG) as a macroinitiator in bulk at 130 degrees C using SnOct(2) as catalyst and by subsequent catalytic hydrogenation. The copolymer could form micelles in aqueous solution with the cmc dependent on the composition of the copolymer. The micelles exhibited a homogeneous spherical morphology and a unimodal size distribution. Their degradation rate in the presence of proteinase K was faster than that of PLA, and they showed a low degree of cytotoxicity to the articular cartilage cells. This biodegradable amphiphilic block copolymer with pendant carboxyl groups is capable of further modification and is expected to facilitate a variety of potential biomedical applications, such as drug carriers, tissue engineering, etc.     

Enhanced stability of polymeric micelles based on postfunctionalized poly(ethylene glycol)-b-poly(γ-propargyl L-glutamate): the substituent effect

One of the major obstacles that delay the clinical translation of polymeric micelle drug delivery systems is whether these self-assembled micelles can retain their integrity in blood following intravenous (IV) injection. The objective of this study was to evaluate the impact of core functionalization on the thermodynamic and kinetic stability of polymeric micelles. The combination of ring-opening polymerization of N-carboxyanhydride (NCA) with highly efficient "click" coupling has enabled easy and quick access to a family of poly(ethylene glycol)-block-poly(γ-R-glutamate)s with exactly the same block lengths, for which the substituent "R" is tuned. The structures of these copolymers were carefully characterized by (1)H NMR, FT-IR, and GPC. When pyrene is used as the fluorescence probe, the critical micelle concentrations (CMCs) of these polymers were found to be in the range of 10(-7)-10(-6) M, which indicates good thermodynamic stability for the self-assembled micelles. The incorporation of polar side groups in the micelle core leads to high CMC values; however, micelles prepared from these copolymers are kinetically more stable in the presence of serum and upon SDS disturbance. It was also observed that these polymers could effectively encapsulate paclitaxel (PTX) as a model anticancer drug, and the micelles possessing better kinetic stability showed better suppression of the initial "burst" release and exhibited more sustained release of PTX. These PTX-loaded micelles exerted comparable cytotoxicity against HeLa cells as the clinically approved Cremophor PTX formulation, while the block copolymers showed much lower toxicity compared to the cremophor-ethanol mixture. The present work demonstrated that the PEG-b-PPLG can be a uniform block copolymer platform toward development of polymeric micelle delivery systems for different drugs through the facile modification of the PPLG block.     

Epidermal growth factor-conjugated poly(ethylene glycol)-block- poly(delta-valerolactone) copolymer micelles for targeted delivery of chemotherapeutics

Epidermal growth factor (EGF)-conjugated copolymer micelles were prepared from a mixture of diblock copolymers of methoxy poly(ethylene glycol)-block-poly(delta-valerolactone) (MePEG-b-PVL) and EGF-PEG-b-PVL for targeted delivery to EGF receptor (EGFR)-overexpressing cancers. The block copolymers and functionalized block copolymers were synthesized using PEG as the macroinitiator and HCl-diethyl ether as the catalyst. The MePEG-b-PVL and the carboxyl-terminated PEG-b-PVL (HOOC-PEG-b-PVL) copolymers were found to have molecular weights of 5940 and 5900, respectively, as determined by gel permeation chromatography (GPC) analyses. The HOOC-PEG-b-PVL copolymers were then activated by N-hydroxysuccinimide and subsequently reacted with EGF to form the EGF-PEG-b-PVL copolymers. The efficiency for the conjugation of EGF to the copolymer was found to be 60.9%. A hydrophobic fluorescent probe, CM-DiI, was loaded into both the nontargeted MePEG-b-PVL micelles and the targeted EGF-conjugated PEG-b-PVL micelles. The effective mean diameters of the CMDiI-loaded nontargeted and the CMDiI-loaded targeted micelles were found to be 32 +/- 1 nm and 45 +/- 2 nm, respectively, as determined by dynamic light scattering (DLS). The zeta potentials for the nontargeted micelles (no CM-DiI-loaded), CM-DiI-loaded nontargeted micelles, and CM-DiI-loaded targeted micelles were found to be -6.5, -8.7, and - 13.5 mV, respectively. Evaluation of the in vitro release of CM-DiI from the MePEG-b-PVL micelles in phosphate buffer saline (0.01 M, pH = 7.4) containing 10% (v/v) fetal bovine serum at 37 degrees C revealed that approximately 20% of the probe was released within the first 2 h. Confocal laser scanning microscopy (CLSM) analysis revealed that the targeted micelles containing CM-DiI accumulated intracellularly in EGFR-overexpressing MDA-MB-468 breast cancer cells following a 2 h incubation period, while no detectable cell uptake was observed for the nontargeted micelles. Results obtained from the confocal images were confirmed in an independent study by measuring the intracellular CM-DiI fluorescence in cell lysate. In addition, the presence of free EGF was found to decrease the extent of uptake of the targeted micelles. Nuclear staining of the cells with Hoechst 33258 indicated that the targeted micelles mainly localized in the perinuclear region and some of the micelles were localized in the nucleus. These results demonstrate that the EGF-conjugated copolymer micelles developed in this study have potential as vehicles for targeting hydrophobic drugs to EGFR-overexpressing cancers.     

Self-assembly of polypeptide-containing ABC-type triblock copolymers in aqueous solution and its pH dependence

Self-assembling of novel biodegradable ABC-type triblock copolymer poly(ethylene glycol)-poly(L-lactide)-poly(L-glutamic acid) (PEG-PLLA-PLGA) is studied. In aqueous media, it self-assembles into a spherical micelle with the hydrophobic PLLA segment in the core and the two hydrophilic segments PEG and PLGA in the shell. With the lengths of PEG and PLLA blocks fixed, the diameter of the micelles depends on the length of the PLGA block and on the volume ratio of H(2)O/dimethylformamide (DMF) in the media. When the PLGA block is long enough, morphology of the self-assembly is pH-dependent. It assembles into the spherical micelle in aqueous media at pH 4.5 and into the connected rod at or below pH 3.2. The critical micelle concentration (cmc) of the copolymer changes accordingly with decreasing solution pH. Both aggregation states can convert to each other at the proper pH value. This reversibility is ascribed to the dissociation and neutralization of the COOH groups in the LGA residues. When the PLGA block is short compared to the PEG or PLLA block, it assembles only into the spherical micelle at various pH values.     

Synthesis and micellization of amphiphilic brush-coil block copolymer based on poly(epsilon-caprolactone) and PEGylated polyphosphoester

A novel biodegradable amphiphilic brush-coil block copolymer consisting of poly(epsilon-caprolactone) and PEGylated polyphosphoester was synthesized by ring opening polymerization. The composition and structure of the copolymer were characterized by 1H NMR, 13C NMR, and FT-IR, and the molecular weight and molecular weight distribution were analyzed by gel permeation chromatograph (GPC) measurements to confirm the diblock structure. These amphiphilic copolymers formed micellar structures in water, and the critical micelle concentrations (CMCs) were around 10(-3) mg/mL, which was determined using pyrene as a fluorescence probe. Transmission electron microscopy (TEM) images showed that the micelles took an approximately spherical shape with core-shell structure, which was further demonstrated by laser light scattering (LLS) technique. The degradation behavior of the polymeric micelle was also investigated in the presence of Pseudomonas lipase and characterized by GPC measurement. Such polymer micelles from brush-coil block copolymers are expected to have wide utility in the field of drug delivery.     

"Schizophrenic" micellization associated with coil-to-helix transitions based on polypeptide hybrid double hydrophilic rod-coil diblock copolymer

A polypeptide hybrid double hydrophilic diblock copolymer (DHBC), poly( N-isopropylacrylamide)- b-poly( l-glutamic acid) (PNIPAM- b-PLGA), was synthesized via the ring-opening polymerization of gamma-benzyl- l-glutamate N-carboxyanhydride (BLG-NCA) using monoamino-terminated PNIPAM as the macroinitiator, followed by deprotection of benzyl groups under alkaline conditions. Containing a thermoresponsive PNIPAM block and a pH-responsive PLGA block, the obtained polypeptide hybrid diblock copolymer molecularly dissolves in aqueous solution at alkaline pH and room temperature but supramolecularly self-assembles into PNIPAM-core micelles at alkaline pH and elevated temperatures and PLGA-core micelles at acidic pH and room temperature accompanied with coil-to-helix transition of the PLGA sequence. The pH- and thermoresponsive "schizophrenic" micellization behavior of PNIPAM- b-PLGA diblock copolymer has been investigated by (1)H NMR, optical transmittance, fluorescence probe measurement, transmission electron microscopy (TEM), dynamic and static laser light scattering (LLS), and circular dichroism (CD) spectroscopy. Moreover, the micellization process was investigated employing stopped-flow light scattering technique. The pH-induced micelle growth of PNIPAM- b-PLGA in aqueous solution exhibits drastically different kinetics compared to that of conventional pH-responsive DHBCs, probably due to the stabilization effects exerted by the formed alpha-helix secondary structures within the PLGA core at low pH. Exhibiting "schizophrenic" micellization, the polypeptide sequence of PNIPAM- b-PLGA can either locate within micelle cores or stabilizing coronas. The incorporation of polypeptide block into DHBCs can endow them with structural versatility, tunable spatial arrangement of chain segments within self-assembled nanostructures, and broader applications in the field of biomedicines.     

Emission spectra from copper proteins containing type-3 centres

Aqueous solutions of copper-proteins containing type-3 centres (ceruloplasmin, tyrosinase, haemocyanin), excited within their absorption bands at 325-345 nm, show typical luminescence spectra. The emission bands peak at 415-445 nm and their decay time is no longer than 10 ns. A strong analogous fluorescence is obtained also by excitation of concentrated solutions of carboxylic acids and amino acids, which show again absorption bands around 330 nm. Such a fluorescence, although less intense, is also observed in copper(II) carboxylate solutions. In contrast, no fluorescence has been recorded in solutions of acetic anhydride and of polypeptides (valinomycin, gramicidin D), which do not have free carboxyl groups. We tentatively attribute this novel fluorescence in the investigated copper proteins to interactions between carboxyl groups of amino acids at, or near, the active site.     

Biodegradable and biocompatible multi-arm star amphiphilic block copolymer as a carrier for hydrophobic drug delivery

Multi-arm star amphiphilic block copolymers (SABCs) with approximately 32 arms were synthesized and characterized for drug delivery applications. A hyperbranched polyester, boltorn^® H40 (H40), was used as the macroinitiator for the ring-opening polymerization of ?-caprolactone (?-CL). The resulting multi-arm H40-poly(?-caprolactone) (H40-PCL-OH) was further reacted with carboxyl terminated methoxy poly(ethylene glycol) (MPEG-COOH) to form H40-PCL-b-MPEG copolymers. The resulting SABCs were characterized by ¹H NMR spectroscopy and gel permeation chromatography (GPC). The critical aggregation concentration (CAC) of H40-PCL-b-MPEG was 3.8 mg/L as determined by fluorescence spectrophotometry. Below the CAC, stable unimolecular micelles were formed with an average diameter of 18 nm as measured by TEM. Above the CAC, unimolecular micelles exhibited agglomeration with an average diameter of 98 nm. The hydrodynamic diameter of these agglomerates was found to be 122 nm, as measured by dynamic light scattering (DLS). The drug loading efficacy of the H40-PCL-b-MPEG micelles was 26 wt%. Drug release study showed an initial burst followed by a sustained release of the entrapped hydrophobic model drug, 5-fluorouracil, over a period of 9–140 h. These results indicate that the H40-PCL-b-MPEG micelles have great potential as hydrophobic drug delivery carriers.     

Functionalized, probe-containing, latex nanospheres.

Synthesis of surface-functionalized, probe-containing latex nanospheres is described. Approximately 40,000 probe ions may be encapsulated in a nanosphere of 50 nm diameter. The probe may be a radionuclide or a lanthanide with long-lived fluorescence. Alternatively, a "cargo" of pharmaceutical interest may be used. The surface of each nanosphere contains thousands of acid groups which may be functionalized for subsequent attachment to biomolecules such as antibodies. Functionalized nanospheres have been successfully coupled to a tobacco virus.     

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.     

Fabrication of multiresponsive shell cross-linked micelles possessing pH-controllable core swellability and thermo-tunable corona permeability

A double hydrophilic ABC triblock copolymer, poly(2-(diethylamino)ethyl methacrylate)-b-poly(2-(dimethylamino)ethyl methacrylate)-b-poly(N-isopropylacrylamide) (PDEA-b-PDMA-b-PNIPAM), containing the well-known pH-responsive PDEA block and thermoresponsive PNIPAM block, was synthesized by atom transfer radical polymerization via sequential monomer addition using ethyl 2-chloropropionate as the initiator. The obtained triblock copolymer exhibits interesting "schizophrenic" micellization behavior in aqueous solution, and supramolecularly self-assembles into three-layer "onion-like" PNIPAM-core micelles at acidic pH's and elevated temperatures and PDEA-core micelles with "inverted" structures at alkaline pH's and room temperature. In both cases, dynamic laser light scattering (LLS) and optical transmittance reveal the presence of near-monodisperse micelles, and the micelle formation/inversion process is fully reversible. Novel shell cross-linked (SCL) micelles with pH-responsive PDEA cores and thermoresponsive PNIPAM coronas were then facilely fabricated from the PDEA-b-PDMA-b-PNIPAM triblock copolymer by cross-linking the PDMA inner shells with 1,2-bis(2-iodoethoxy)ethane. The reversible pH-dependent swelling/shrinking of PDEA cores and thermosensitive collapse/aggregation of PNIPAM coronas of the obtained SCL micelles were investigated in detail by dynamic LLS, optical transmittance, and transmission electron microscopy. As the structurally stable SCL micelles possess pH-controllable core swellability and thermo-tunable corona permeability, the release profile of a model hydrophobic drug, dipyridamole, initially loaded within the hydrophobic PDEA core, can be dually controlled by both the solution pH and the temperature. This represents the first report of SCL micelles with multiresponsive cores and coronas, which may find practical applications in fields such as drug delivery and smart release.     

Tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.     

Cytotoxicity Study on Luminescent Nanocrystals Containing Phospholipid Micelles in Primary Cultures of Rat Astrocytes

Luminescent colloidal nanocrystals (NCs) are emerging as a new tool in neuroscience field, representing superior optical probes for cellular imaging and medical diagnosis of neurological disorders with respect to organic fluorophores. However, only a limited number of studies have, so far, explored NC applications in primary neurons, glia and related cells. Indeed astrocytes, as resident cells in the central nervous system (CNS), play an important pathogenic role in several neurodegenerative and neuroinflammatory diseases, therefore enhanced imaging tools for their thorough investigation are strongly amenable. Here, a comprehensive and systematic study on the in vitro toxicological effect of core-shell type luminescent CdSe@ZnS NCs incorporated in polyethylene glycol (PEG) terminated phospholipid micelles on primary cultures of rat astrocytes was carried out. Cytotoxicity response of empty micelles based on PEG modified phospholipids was compared to that of their NC containing counterpart, in order to investigate the effect on cell viability of both inorganic NCs and micelles protecting NC surface. Furthermore, since the surface charge and chemistry influence cell interaction and toxicity, effect of two different functional groups terminating PEG-modified phospholipid micelles, namely amine and carboxyl group, respectively, was evaluated against bare micelles, showing that carboxyl group was less toxic. The ability of PEG-lipid micelles to be internalized into the cells was qualitatively and quantitatively assessed by fluorescence microscopy and photoluminescence (PL) assay. The results of the experiments clearly demonstrate that, once incorporated into the micelles, a low, not toxic, concentration of NCs is sufficient to be distinctly detected within cells. The overall study provides essential indications to define the optimal experimental conditions to effectively and profitably use the proposed luminescent colloidal NCs as optical probe for future in vivo experiments.     

Comb polymers prepared by ATRP from hydroxypropyl cellulose

Hydroxypropyl cellulose (HPC) was used as a core molecule for controlled grafting of monomers by ATRP, the aim being to produce densely grafted comb polymers. HPC was either allowed to react with an ATRP initiator or the first generation initiator-functionalized 2,2-bis(methylol)propionic acid dendron to create macroinitiators having high degrees of functionality. The macroinitiators were then "grafted from" using ATRP of methyl methacrylate (MMA) or hexadecyl methacrylate. Block copolymers were obtained by chain extending PMMA-grafted HPCs via the ATRP of tert-butyl acrylate. Subsequent selective acidolysis of the tert-butyl ester moieties was performed to form a block of poly(acrylic acid) resulting in amphiphilic block copolymer grafts. The graft copolymers were characterized by 1H NMR and FT-IR spectroscopies, DSC, TGA, rheological measurements, DLS, and tapping mode AFM on samples spin coated upon mica. It was found that the comb (co)polymers were in the nanometer size range and that the dendronization had an interesting effect on the rheological properties.     

Polymeric micelle-based bioassay with femtomolar sensitivity

Target-specific polymeric micelles loaded with fluorescence dye molecules in their hydrophobic cores were made from block copolymer of poly(caprolactones)23-b-poly(ethylene oxide)45. It was found that the micelles are stable against pH changes from pH 2 to 12 and temperature variation up to 65 degrees C. The dye molecules can be released to the solution on exposing the micelles to organic solvents or ultrasound. A rapid and highly sensitive immunoassay based on the above micelles was developed, and the assay can detect specific target proteins in the femtomolar range from complex biological samples such as serum mimics and cell lysate. For example, less than 0.15 U/ml of ovarian cancer-specific antigen 125, equivalent to 7.5 x 10(-15)M, can be reliably detected in solution. We also demonstrated that the assay can detect a cell surface biomarker, stage-specific embryonic antigen 4, from a single human embryonic stem cell.     

Enhanced stability of core-surface cross-linked micelles fabricated from amphiphilic brush copolymers

"Stealth" nanoparticles made from polymer micelles have been widely explored as drug carriers for targeted drug delivery. High stability (i.e., low critical micelle concentration (CMC)) is required for their intravenous applications. Herein, we present a "core-surface cross-linking" concept to greatly enhance nanoparticle's stability: amphiphilic brush copolymers form core-surface cross-linked micelles (nanoparticles) (SCNs). The amphiphilic brush copolymers consisted of hydrophobic poly(epsilon-caprolactone) (PCL) and hydrophilic poly(ethylene glycol) (PEG) or poly(2-(N,N-dimethylamino)ethyl methacrylate) (PDMA) chains were synthesized by macromonomer copolymerization method and used to demonstrate this concept. The resulting SCNs were about 100 times more stable than micelles from corresponding amphiphilic block copolymers. The size and surface properties of the SCNs could be easily tailored by the copolymer's compositions.     

Membrane proteins in reverse micelles: myelin basic protein in a membrane-mimetic environment

The solubility, reactivity, and conformational dynamics of myelin basic protein (MBP) from bovine brain were studied in reverse micelles of sodium bis(2-ethylhexyl) sulfosuccinate (AOT)-isooctane and water. Such a membrane-mimetic system resembles the aqueous spaces of native myelin sheath in terms of physicochemical properties as reflected in the high affinity of MBP for interfacial bound water. This is marked by the unusual profile of the solubility curve of the protein in reverse micelles, which shows optimal solubility at a much lower molar ratio of water to surfactant ([ H2O]/[AOT] = w0) than that reported for other water-soluble proteins. The role of counterions and/or charged polar head groups in the solubilization process is revealed by comparison of the solubility of MBP in nonionic surfactant micellar solutions. Whereas MBP is unfolded in aqueous solutions, insertion into reverse micelles generates a more folded structure, characterized by the presence of 20% alpha-helix. This conformation is unaffected by variations in the water content of the system (in the 2.0-22.4 w0 range). The reactivity of epsilon-amino groups of lysine residues with aqueous solutions of o-phthalaldehyde demonstrates that segments of the peptide chain are accessible to water. Similar results were obtained with the sequence involved in heme binding. In contrast, the sole tryptophan residue, Trp-117, is shielded from the aqueous solvent, as indicated by lack of reaction with N-bromosuccinimide. The invariance of the wavelength maximum emission in the fluorescence spectra as a function of w0 is consistent with this result.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization and controlled release of a hydrophobic drug using novel micelle-forming ABC triblock copolymers

Amphiphilic ABC triblock copolymers composed of monomethoxy-capped poly(ethylene glycol) (MPEG), poly(2-(dimethylamino)ethyl methacrylate) (DMA), and poly(2-(diethylamino)ethyl methacrylate) (DEA) have been synthesized by atom transfer radical polymerization (ATRP). These copolymers dissolve molecularly in acidic aqueous media at room temperature due to protonation of the tertiary amine groups on the DMA and DEA residues. On adjusting the pH with base, micellization occurred at pH 8, with the water-insoluble, deprotonated DEA block forming the hydrophobic cores and the MPEG and DMA blocks forming the hydrophilic micellar coronas and inner shells, respectively. This pH-induced micellization has been exploited to develop a solvent-free protocol for drug loading. A model hydrophobic drug, dipyridamole (DIP), which dissolves in acid but is insoluble above pH 5.8, was incorporated into the micelles by increasing the pH of an aqueous drug/copolymer mixture to 9. Both the empty and the drug-loaded micelles were characterized by dynamic light scattering and fluorescence studies. The interaction of both pyrene and DIP with the MPEG-DMA-DEA micelles was studied by fluorescence; both compounds had relatively high partition coefficients into the micelles, 4.5 x 10(5) and 1.5 x 10(4), respectively. Intensity-average micelle diameters ranged from 20 to 90 nm, depending on the polymer composition and concentration. Shorter MPEG blocks (Mn = 2000) produced larger micelles than longer MPEG blocks (Mn = 5000) due to the shift in the hydrophilic-hydrophobic balance of the copolymer. Transmission electron microscopy studies of the drug-loaded micelles indicated spherical morphologies and reasonably uniform particle size distributions, which is in marked contrast to the needlelike morphology observed for pure DIP in the absence of the copolymer. Experiments on controlled release demonstrated that DIP-loaded MPEG-DMA-DEA micelles act as a drug carrier, giving slow release to the surrounding solution over a period of days. Rapid release can be triggered by reducing the pH to reverse the micellization.