Heterobifunctional PEG Research Articles

Versatile and Selective Synthesis of “Click Chemistry” Compatible Heterobifunctional Poly(ethylene glycol)s Possessing Azide and Alkyne Functionalities

Versatile route for "click chemistry" compatible heterobifunctional PEGs was established through preparation of alpha-tetrahydropyranyloxy-omega-hydroxyl poly(ethylene glycol) (THP-PEG-OH) via ring-opening polymerization of ethylene oxide using 2-(tetrahydro-2H-pyran-2-yloxy)ethanol as an initiator, followed by the functionalization of omega-OH group to either the azido or alkyne group. Quantitative azidation of THP-PEG-OH was confirmed from the analysis of molecular functionality of the derivatives. While the conversion efficiency of omega-alkynation was appropriately 70%, the unreacted THP-PEG-OH fraction was successfully removed by ion-exchange chromatography after the carboxylation of the hydroxyl group with succinic anhydride. Then, the protecting group of the alpha-end, THP, was removed in mild acidic media, followed by two- or three-step modification of the resulting alpha-hydroxyl group to primary amino or thiol groups. Consequently, "click chemistry" compatible heterobifunctional PEG derivatives (X-PEG-Y; X = NH(2) and SH, Y =Azide and Alkyne) were synthesized with high efficiency and controlled molecular weight.

Synthesis of heterobifunctional poly(ethylene glycol)s by an acetal protection method

A versatile synthetic route to prepare well-defined heterobifunctional poly(ethylene glycol) (PEG) derivatives via sequential modification of the hydroxyl ends was established using a newly synthesized initiator for anionic polymerization, 2-(1-ethoxyethoxy)ethanol (EEE), which contains an acid-cleavable acetal protection group. As illustrative examples, two heterobifuntional PEGs, HO–PEG–alkyne and HS–PEG–alkyne, were successfully prepared. This approach is applicable for synthesizing a wide variety of heterobifunctional PEGs.

Integrin ανβ3 Targeted Gene Delivery Using RGD Peptidomimetic Conjugates with Copolymers of PEGylated Poly(ethylene imine)

This study describes the synthesis and characterization of five conjugates of poly(ethylene glycol) modified polyethylenimine (PEG-PEIs) coupled in two different synthesis routes to a nonpeptidic pentacyclic RDG-mimetic for integrin receptor-targeted gene delivery. Synthesis of this panel of different conjugates allowed for systematic analysis of structure-activity relationships. Conjugates were therefore characterized regarding molecular composition, DNA condensation, size, and zeta potential of self-assembled polyplexes. In vitro characterization included investigation of blood compatibility, binding affinity to receptor-positive and receptor-negative cells measured by flow cytometry, cellular uptake quantified by scintillation counting, and efficiency and specificity of transfection assayed by reporter gene expression. In a first synthetic approach, low molecular weight PEI (LMW-PEI) was PEGylated using a heterobifunctional PEG linker and coupling of the RGD-mimetic was achieved at the distal end of PEG chains. In a second synthesis route, the RGD-mimetic was directly coupled to AB-block-copolymers of PEI (25 kDa) and PEG (30 kDa). Interactions of RGD-PEG-LMW-PEI conjugates with DNA were strongly impaired, whereas PEG-PEI-RGD conjugates were more promising candidates due to their physicochemical properties and higher receptor specificity. The binding, uptake, and transfection efficiency in receptor-positive cells was strongly increased upon conjugation of the RGD-mimetic to AB-block-copolymers of PEG-PEI and depended on the degree of peptide substitution. The conjugates of PEG-PEI AB-block-copolymers with low ligand density of the RGD-mimetic appear to be promising candidates for in vivo cancer gene therapy.

Vinyl Sulfone-Terminated PEG−PLLA Diblock Copolymer for Thiol-Reactive Polymeric Micelle

Vinyl sulfone-terminated polyethylene glycol-poly(l-lactide) (VS−PEG−PLLA) diblock copolymer was designed as an amphiphilic structure containing a VS group with high reactivity toward thiols. Prior to the copolymer synthesis, heterobifunctional PEG with a VS group at one terminus was prepared and then PLLA was introduced to the other end of PEG. The chemical structure and molecular weight of the resulting products was assigned by 1H NMR and GPC. As micellar properties of VS−PEG−PLLA under aqueous environment, the critical micelle concentration and mean size of VS−PEG−PLLA were found to be approximately 59.8 mg/L and 34 nm, respectively. Kinetic studies of the VS-functionalized PEG−PLLA micelle showed that the reaction rates were affected by various factors but most of cysteine-contained peptides reacted rapidly within 1 h via Michael-type addition. Therefore, this thiol-reactive polymeric micelle is expected to be very useful for a functional nanocarrier system through the effective conjugation of cystein...

Design of Core−Shell-Type Nanoparticles Carrying Stable Radicals in the Core

Utilizing the self-assembled core-shell-type polymeric micelle technique, high-performance nanoparticles possessing stable radicals in the core and reactive groups on the periphery were prepared. The anionic ring-opening polymerization of ethylene oxide (EO) was carried out using potassium 3,3-diethoxypropanolate as an initiator, followed by mesylation with methanesulfonyl chloride to obtain acetal-poly(ethylene glycol)-methanesulfonate (acetal-PEG-Ms; 1). Compound 1 was reacted with potassium O-ethyldithiocarbonate, followed by treatment with n-propylamine to obtain heterobifunctional PEG derivatives containing both sulfanyl and acetal terminal groups (acetal-PEG-SH) (2) in a highly selective and quantitative manner. Poly(ethylene glycol)-block-poly(chloromethylstyrene) (acetal-PEG-b-PCMS) (3) was synthesized by the free-radical telomerization of chloromethylstyrene (CMS) using 2 as a telogen. The chloromethyl groups in the PCMS segment of the block copolymer (3) were quantitatively converted to 2,2,6,6-tetramethylpiperidinyloxys (TEMPOs) via the amination of 3 with 4-amino-TEMPO to obtain acetal-PEG-b-PCMS containing TEMPO moieties (4). The obtained 4 formed core-shell-type nanoparticles in aqueous media when subjected to the dialysis method: the cumulant average diameter of the nanoparticles was about 40 nm, and the nanoparticles emitted intense electron paramagnetic resonance (EPR) signals. The TEMPO radicals in the core of the nanoparticles showed reduction resistance even in the presence of 3.5 mM ascorbic acid. This means that these nanoparticles are anticipated as high-performance bionanoparticles that can be used in vivo.

Efficient Catalytic Synthesis of Dendritic Polymers Having Internal Fluorescence Labels for Bioconjugation

Here we present an efficient synthesis of functional dendritic polymers carrying internal fluorescence labels for bioconjugation. Specifically, dendritic polymers having pyrene as fluorescence label in the core and N-hydroxysuccinimide (NHS) functional groups at the periphery were synthesized by coupling heterobifunctional PEG to hydroxyl functionalized dendritic polyethylene core. The dendritic polyethylene cores containing one pyrene label per polymer molecule were prepared through a one-step transition-metal-catalyzed polymerization using a pyrene-labeled Pd(II)-alpha-diimine chain walking catalyst. A series of pyrene-labeled dendritic scaffolds were obtained with different molecular weights and sizes. NHS active end groups were introduced to the periphery of the dendritic scaffolds through end-group functionalization. Those NHS-functionalized dendritic scaffolds were successfully used to conjugate a model protein, ovalbumin, to yield protein-polymer conjugates carrying multiple copies of protein attached to each scaffold.

Antitumoral activity of PEG–gemcitabine prodrugs targeted by folic acid

Gemcitabine, 2′,2′-difluoro-2′-deoxycytidine (dFdC), is an antitumor agent effective in the treatment of several solid tumors but its use is hampered by short plasma half-life, rapid metabolism and low selectivity towards tumor tissue. To overcome these limits, bioconjugates of gemcitabine were studied using poly(ethylene glycol) as polymeric carrier. Two types of conjugates were prepared, non-targeted and folic acid targeted conjugates. The formers were obtained starting from mPEG–OH of 5 and 20 kDa with linear or branched structure. The folic acid targeted conjugates, differing for the drug loading, were prepared exploiting a heterobifunctional PEG that allowed a consecutive coupling of the targeting agent and the drug. Folic acid was chosen as targeting agent because its receptor is often over-expressed in many tumors. To increase the polymer drug payload, the bicarboxylic amino acid, aminoadipic acid, was used. All conjugates were able to release the drug in a pH-dependent manner with no role of enzymes. The pharmacokinetic profiles are strictly related to the polymer molecular weight and the folic acid targeting increased 2–3 times the affinity towards the cells over-expressing folic acid receptors. These results are promising and encourage in vivo studies on these conjugates that act as polymeric prodrugs.

An efficient mixed solid–liquid phase synthesis of a heterobifunctional amphiphilic PEG–NH 2 derivative and its conjugation to folic acid

A very efficient, versatile as well as simple to perform procedure was developed in order to prepare a heterobifunctional amphiphilic PEG–NH 2 derivative which can be used for conjugation to a targeting ligand (such as folic acid). This method proceeds by a mixed solid–liquid phase strategy using a TentaGel ® PAP resin, a copolymer consisting of a polystyrene matrix on which a PEG ( M w 3400 Da) terminated by an amino function has been grafted. Solid phase chemistry was used for the conjugation of a highly hydrophobic moiety. After release from the resin, the amphiphilic PEG–OH conjugate was converted into its corresponding amphiphilic PEG–NH 2 derivative (four steps in 77% overall yields). This procedure allowed the preparation of ∼330 mg batches. This derivative was then coupled to folic acid, a ligand that is used for the targeting of drug (gene) carrier and delivery systems to cells over-expressing the folate receptor. The low and high molecular weight of folic acid and its amphiphilic PEG–folate conjugate, respectively, allowed easy purification by dialysis and led to the targeted compound with high recovery.

A New, Simple Method for Linking of Antibodies to Atomic Force Microscopy Tips

Functionalization of atomic force microscope (AFM) tips with bioligands converts them into monomolecular biosensors which can detect complementary receptor molecules on the sample surface. Flexible PEG tethers are preferred because the bioligand can freely reorient and locally palpate the sample surface while the AFM tip is moved along. In a well-established coupling scheme [Hinterdorfer et al. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 3477-3481], a heterobifunctional PEG linker is used to tether thiol-containing bioligands to amino-functionalized AFM tips. Since antibodies contain no free thiol residues, prederivatization with N-succinimidyl 3-(acetylthio)propionate (SATP) is needed which causes a relatively high demand for antibody. The present study offers a convenient alternative with minimal protein consumption (e.g., 5 microg of protein in 50 microL of buffer) and no prederivatization, using a new heterobifunctional cross-linker that has two different amino-reactive functions. One end is an activated carboxyl (N-hydroxysuccinimide ester) which is much faster to react with the amino groups of the tips than the benzaldehyde function on its other end. The reactivity of the latter is sufficient, however, to covalently bind lysine residues of proteins via Schiff base formation. The method has been critically examined, using biotinylated IgG as bioligand on the tip and mica-bound avidin as complementary receptor. These experiments were well reproduced on amino-functionalized silicon nitride chips where the number of specifically bound IgG molecules (approximately 2000 per microm2) was estimated from the amount of specifically bound ExtrAvidin-peroxidase conjugate. For a bioscientific application, human rhinovirus particles were tethered to the tip, very-low-density lipoprotein receptor fragments were tethered to mica, and the specific interaction was studied by force microscopy.

Antibody Linking to Atomic Force Microscope Tips via Disulfide Bond Formation

Covalent binding of bioligands to atomic force microscope (AFM) tips converts them into monomolecular biosensors by which cognate receptors can be localized on the sample surface and fine details of ligand-receptor interaction can be studied. Tethering of the bioligand to the AFM tip via a approximately 6 nm long, flexible poly(ethylene glycol) linker (PEG) allows the bioligand to freely reorient and to rapidly "scan" a large surface area while the tip is at or near the sample surface. In the standard coupling scheme, amino groups are first generated on the AFM tip. In the second step, these amino groups react with the amino-reactive ends of heterobifunctional PEG linkers. In the third step, the 2-pyridyl-S-S groups on the free ends of the PEG chains react with protein thiol groups to give stable disulfide bonds. In the present study, this standard coupling scheme has been critically examined, using biotinylated IgG with free thiols as the bioligand. AFM tips with PEG-tethered biotin-IgG were specifically recognized by avidin molecules that had been adsorbed to mica surfaces. The unbinding force distribution showed three maxima that reflected simultaneous unbinding of 1, 2, or 3 IgG-linked biotin residues from the avidin monolayer. The coupling scheme was well-reproduced on amino-functionalized silicon nitride chips, and the number of covalently bound biotin-IgG per microm2 was estimated by the amount of specifically bound ExtrAvidin-peroxidase conjugate. Coupling was evidently via disulfide bonds, since only biotin-IgG with free thiol groups was bound to the chips. The mechanism of protein thiol coupling to 2-pyridyl-S-S-PEG linkers on AFM tips was further examined by staging the coupling step in bulk solution and monitoring turnover by release of 2-pyridyl-SH which tautomerizes to 2-thiopyridone and absorbs light at 343 nm. These experiments predicted 10(3)-fold slower rates for the disulfide coupling step than actually observed on AFM tips and silicon nitride chips. The discrepancy was reconciled by assuming 10(3)-fold enrichment of protein on AFM tips via preadsorption, as is known to occur on comparable inorganic surfaces.

Site‐Specific Immobilization of Genetically Engineered Variants of Candida antarctica Lipase B

The immobilization of proteins on solid surfaces has been a topicof intensive researc h for many years. Numerous methods have been developed to immobilize proteins for bioseparation, biosensors, diagnostictests and single-molec ule experiments. [1] With the growing need for miniaturization and parallelization, new methods are needed for the immobilization of proteins with high functional density and specificity. [2, 3] Most of the methods used so far do not allow the covalent immobilization of a protein at a well-defined position. In this study, cysteines, introduced by genetic engineering, have been used for site-specific immobilization of a model enzyme to a glass surface by means of a heterobifunctional poly(ethylene glycol) (PEG) spacer. [4] While PEG is used for a broad range of applications to render surfaces protein resistant, [5–7] only a few reports describe its use as a linker for attaching proteins to surfaces. [8–13] However, the use of a heterobifunctional PEG spacer would provide a protein-resistant surface displaying reactive groups for the covalent attachment of proteins in a controlled manner. In addition, the coupling of PEG to proteins has been shown to increase the stability of the protein and maintain it in an active conformation. [14] To evaluate the usefulness of this concept, Candida antarctica lipase B (CalB; EC 3.1.1.3) was used as a model enzyme. [15] CalB is an industrially important lipase that is used for various applications in bioorganic synthesis. [16] It is a 35 kDa monomer with three disulfide bridges. CalB has been immobilized on surfaces for various applications, but these methods are mainly based on the adsorption of hydrophobicor c harged amino acids to supports. [17] Site-specific immobilization of lipases would be advantageous because it has been shown that the properties of lipases depend on their orientation on a surface. [18] In this study, mutants of CalB have been prepared that display a free cysteine at a defined position on the molecule

Immobilization of heparin on a silicone surface through a heterobifunctional PEG spacer

A novel method of immobilizing heparin on a silicone surface through a heterobifunctional PEG spacer was used yield well defined surfaces with highly active surface immobilized heparin and low non-specific protein adsorption. The heparin surface density achieved using this technique was 0.68 μg/cm 2. Sessile drop water contact angles showed increased hydrophilicity of the silicone surface after PEG modification and a further decrease in the contact angles following the grafting of heparin. High specificity for ATIII with little fibrinogen adsorption was noted in plasma adsorption studies. This ATIII adsorption was mediated by the heparin layer, since surfaces modified with PEG only did not adsorb significant quantities of AT. The thrombin resistance of the heparin modified surfaces was demonstrably greater as measured by a chromogenic thrombin generation assay. The results suggest that the heterbifunctional PEG linker results in a high density of active heparin on the surfaces.

A Reactive Poly(ethylene glycol) Layer To Achieve Specific Surface Plasmon Resonance Sensing with a High S/N Ratio: The Substantial Role of a Short Underbrushed PEG Layer in Minimizing Nonspecific Adsorption

A reactive poly(ethylene glycol) (PEG)-brushed layer was constructed on a surface plasmon resonance (SPR) sensor chip using a heterobifunctional PEG possessing an acetal group at one end and a mercapto group at the other end (alpha-acetal-omega-mercapto-PEG). The density of the PEG brushed layer substantially increased with repetitive adsorption/rinse cycles of the PEG on the sensor chip, allowing dramatic reduction of nonspecific protein adsorption. Notably, formation of a short, filler layer of PEG (2 kDa) in the preconstructed longer PEG brushed layer (5 kDa) achieved almost complete prevention of nonspecific protein adsorption. The acetal group located at the distal end of the tethered PEG was converted to an aldehyde group by the acid treatment, followed by the installation of biocytin hydrazide through Schiff base formation. SPR sensing of streptavidin was done with a very high S/N ratio even in a proteinous medium using the biotinylated PEG (5 kDa) tethered chip with an inert filler layer of short PEG (2 kDa). Furthermore, the specific affinity of streptavidin for the biotinylated PEG was highly influenced by the length of the filler PEG and was significantly reduced when the length of the filler PEG was longer than that of the biotinylated PEG. This result clearly revealed the substantial importance of the steric factor on biospecific interaction at the distal end of tethered PEG on the sensor surface.

Heterobifunctional crosslinkers for tethering single ligand molecules to scanning probes

Single molecule recognition force microscopy (SMRFM) is a versatile atomic force microscopy (AFM) method to probe specific interactions of cognitive molecules on the single molecule level. It allows insights to be gained into interaction potentials and kinetic barriers and is capable of mapping interaction sites with nm positional accuracy. These applications require a ligand to be attached to the AFM tip, preferably by a distensible poly(ethylene glycol) (PEG) chain between the measuring tip and the ligand molecule. The PEG chain greatly facilitates specific binding of the ligand to immobile receptor sites on the sample surface. The present study contributes to tip-PEG-ligand tethering in three ways: (i) a convenient synthetic route was found to prepare NH 2–PEG–COOH which is the key intermediate for long heterobifunctional crosslinkers; (ii) a variety of heterobifunctional PEG derivatives for tip-PEG-ligand linking were prepared from NH 2–PEG–COOH; (iii) in particular, a new PEG crosslinker with one thiol-reactive end and one terminal nitrilotriacetic acid (NTA) group was synthesized and successfully used to tether His 6-tagged protein molecules to AFM tips via noncovalent NTA–Ni 2+–His 6 bridges. The new crosslinker was applied to link a recombinant His 6-tagged fragment of the very-low density lipoprotein receptor to the AFM tip whereupon specific docking to the capsid of human rhinovirus particles was observed by force microscopy. In a parallel study, the specific interaction of the small GTPase Ran with the nuclear import receptor importin β1 was studied in detail by SMRFM, using the new crosslinker to link His 6-tagged Ran to the measuring tip [Nat. Struct. Biol. (2003), 10, 553–557].

PH-responsive oligodeoxynucleotide (ODN)-poly(ethylene glycol) conjugate through acid-labile beta-thiopropionate linkage: preparation and polyion complex micelle formation.

An oligodeoxynucleotide (ODN) conjugated to poly(ethylene glycol) (PEG) through a pH-responsive ester linkage (PEG-ODN conjugate) was successfully synthesized by the Michael reaction of 3'-thiol-modified ODN with a heterobifunctional PEG bearing an acetal group at the alpha-end and an acrylate group at the omega-end (acetal-PEG-acrylate), aimed at the development of a novel ODN delivery system. The prepared PEG-ODN conjugate and linear-poy(ethyleneimine) (L-PEI) spontaneously associated to form a polyion complex (PIC) micelle whose diameter and polydispersity index micro(2)/Gamma(2)) were 102.5 nm and 0.096 as determined by DLS measurements, respectively. Both the PEG-ODN conjugate and PIC micelle showed cleavage of the ester linkage at the endosomal pH (=5.5), suggesting that the PIC micelle is anticipated to release the ODN in the intracellular compartment. Furthermore, the PEG-ODN conjugate in the PIC micelle was stable against deoxyribonuclase (DNase I) digestion and has no interaction with the serum component because of the steric stabilization of the highly dense PEG corona surrounding the PIC core. These characteristics of the PIC micelles entrapping the PEG-ODN conjugate are promising for their utility as a novel ODN delivery system.

PEGylated nanoparticles for biological and pharmaceutical applications

The utility of polymeric micelles formed through the multimolecular assembly of block copolymer was comprehensively described as novel core–shell typed colloidal carriers for drug and gene targeting. Particularly, novel approaches for the formation of functionalized poly(ethylene glycol) (PEG) layers as hydrophilic outer shell were focused to attain receptor-mediated drug and gene delivery through PEG-conjugated ligands with a minimal non-specific interaction with other proteins. Surface organization of block copolymer micelles with cross-linking core was also described from a standpoint of the preparation of a new functional surface-coating with a unique macromolecular architecture. The micelle-attached surface and the thin hydrogel layer made by layered micelles exhibited nonfouling properties and worked as the reservoir for hydrophobic reagents. Furthermore, the potential utility of multimolecular assembly derived from heterobifunctional PEGs and block copolymers were explored to systematically modify the properties of metal and semiconductor nanostructures by controlling their structure and their surface properties, making them extremely attractive for use in biological and biomedical applications.

Quantitative and reversible lectin-induced association of gold nanoparticles modified with alpha-lactosyl-omega-mercapto-poly(ethylene glycol).

Gold nanoparticles (1-10 nm size range) were prepared with an appreciably narrow size distribution by in situ reduction of HAuCl(4) in the presence of heterobifunctional poly(ethylene glycol) (PEG) derivatives containing both mercapto and acetal groups (alpha-acetal-omega-mercapto-PEG). The alpha-acetal-PEG layers formed on gold nanoparticles impart appreciable stability to the nanoparticles in aqueous solutions with elevated ionic strength and also in serum-containing medium. The PEG acetal terminal group was converted to aldehyde by gentle acid treatment, followed by the reaction with p-aminophenyl-beta-D- lactopyranoside (Lac) in the presence of (CH(3))(2)NHBH(3). Lac-conjugated gold nanoparticles exhibited selective aggregation when exposed to Recinus communis agglutinin (RCA(120)), a bivalent lectin specifically recognizing the beta-D-galactose residue, inducing significant changes in the absorption spectrum with concomitant visible color change from pinkish-red to purple. Aggregation of the Lac-functionalized gold nanoparticles by the RCA(120) lectin was reversible, recovering the original dispersed phase and color by addition of excess galactose. Further, the degree of aggregation was proportional to lectin concentration, allowing the system to be utilized to quantitate lectin concentration with nearly the same sensitivity as ELISA. This simple, yet highly effective, derivatization of gold nanoparticles with heterobifunctional PEG provides a convenient method to construct various colloidal sensor systems currently applied in bioassays and biorecognition.

A Collection of Long, Flexible PEG-Crosslinkers for Molecular Recognition Force Microscopy on Single Molecules

Attachment of a single probe molecule (e.g. an antibody) to the measuring tip of an atomic force microscope yields a powerfull sensor for specific detection of single target molecules. As evidenced by many studies, target recognition by the probe is much facilitated when inserting a poly(ethylene glycol) (PEG) chain (∼8 nm) between probe and tip [1]. This presentation points out future directions of coupling via long, flexible spacers. Starting from highly pure NH2 -PEG-COOH, a variety of heterobifunctional PEG derivatives with amino-, carbonyl-, and different thiol-reactive end groups, as well as with biotin or nitrilotriacetic acid (NTA) termini, were synthesized and applied in recognition force microscopy (Fig. 1, next page) [2]. Tip-bound PEG with an NTA terminus proved useful to “snatch” a His6 -tagged probe (the ectodomain of a virus receptor) to study the interaction with the ligand sites on immobilized viruses. The avidin-biotin interaction was utilized for methodical purposes (Fig. 2, next page). Attachment of biotin-PEG-COOH to aminofunctionalized tips and adsorption of avidin to mica provided for easy model experiments with clear-cut controls that allowed developing new modes of recognition imaging in dynamic force microscopy [3].

Cationic poly(ethyleneglycol) lipids incorporated into pre-formed vesicles enhance binding and uptake to BHK cells

This paper describes a new method for enhancing the interaction of liposomes with cells. A novel class of cationic poly(ethyleneglycol) (PEG)-lipid (CPL) conjugates have been characterized for their ability to insert into pre-formed vesicles and enhance in vitro cellular binding and uptake of neutral and sterically-stabilized liposomes. The CPLs, which consist of a distearoylphosphatidylethanolamine (DSPE) anchor, a fluorescent dansyl moiety, a heterobifunctional PEG polymer ( M r 3400), and a cationic headgroup composed of lysine derivatives, have been described previously [Bioconjug. Chem. 11 (2000) 433]. Five separate CPL, possessing 1–4 positive charges in the headgroup (referred to as CPL 1–CPL 4, respectively), were incubated (as micellar solutions) in the presence of neutral or sterically-stabilized cationic large unilamellar vesicles (LUVs), and were found to insert into the external leaflet of the LUVs in a manner dependent on temperature, time, CPL/lipid ratio, and LUV composition. For CPL/lipid molar ratios ≤0.1, optimal insertion levels of approximately 70% of initial CPL were obtained following 3 h at 60°C. The insertion of CPL resulted in aggregation of the LUVs, as assessed by fluorescence microscopy, which could be prevented by the presence of 40 mM Ca 2+. The effect of CPL-insertion on the binding of LUVs to cells was examined by fluorescence microscopy and quantified by measuring the ratio of rhodamine fluorescence to protein concentration. Neither control LUVs or LUVs containing CPL 2 displayed significant uptake by BHK cells. However, a 3-fold increase in binding was observed for LUVs possessing CPL 3, while for CPL 4-LUVs values as high as 10-fold were achieved. Interestingly, the increase in lipid uptake did not correlate with total surface charge, but rather with increased positive charge density localized at the CPL distal headgroups. These results suggest that incorporation of CPLs into existing liposomal drug delivery systems may lead to significant improvements in intracellular delivery of therapeutic agents.

Polymeric micelles as drug delivery systems: a reactive polymeric micelle carrying aldehyde groups

Nanospheric particles as drug delivery systems are gaining increasing interest in the biomedical field. Nanospheres have been proven as efficient drug delivery systems for intravenous administration because of their comparatively long bloodstream circulation. A novel approach in the field of polymeric drug delivery systems was introduced by the formation of polymeric micelles and subsequently by functionalized polymeric micelles. Functionalized polymeric micelles are expected to find a wide application in the fields of drug delivery and diagnosis since the possibility of coupling to bioactive substances is provided. A large number of densely packed functional groups on the outer shell of the micelle allows an immobilization of biologically active substances at a high density. This is a great advantage for utilizing this particular type of nanosphere in the biomedical field. The possibilities of synthesizing heterobifunctional block copolymers will be demonstrated and the influence of the individual block length on the micelle properties will be discussed. Functionalized polymeric micelles were synthesized from poly(ethylene glycol) (PEG) and poly(lactide) (PLA), and combine the advantages given by the hydrophobic PLA core and the hydrophilic PEG corona. An established quantitative synthetic method for the formation of heterobifunctional PEG was advanced and applied to the block copolymerization. A heterobifunctional block copolymer was synthesized, terminated by an acetal group at the PEG end and a vinyl group was introduced at the PLA end in a one-pot synthesis. After the micellization the acetal groups on the micelle surface were converted into aldehyde groups by an acidic treatment. Aldehyde groups can react rapidly with primary amines forming Schiff bases, a potential future pathway for the conjugation of functionalized polymeric micelles with proteins. PLA was chosen as core-forming segment since it is a biodegradable, non-toxic polymer that is well established as implant material. Dynamic and static light scattering was applied to determine the micelle size and shape and to study the dependence of the micelle geometry on the block length of the copolymer. © 1998 John Wiley & Sons, Ltd.