Novel prodrugs of SN38 using multiarm poly(ethylene glycol) linkers

CPT-11, also known as irinotecan, is a prodrug that is approved for the treatment of advanced colorectal cancer. The active metabolite of CPT-11, SN38 (7-ethyl-10-hydroxy-camptothecin), has 100- to 1000-fold more potent cytotoxic activity in tissue cell culture compared with CPT-11. However, parental administration of SN38 is not possible because of its inherently poor water solubility. It is reported here that a multiarm poly(ethylene glycol) (PEG) backbone linked to four SN38 molecules (PEG-SN38) has been successfully prepared with high drug loading and significantly improved water solubility (400- to 1000-fold increase). Three different protecting strategies have been developed in order to selectively acylate the 20-OH of SN38 to preserve its E-ring in the lactone form (the active form of SN38 with cytotoxic activities) while PEG is still attached. One chemical process has been optimized to make a large quantity of the PEG-SN38 conjugate with a high yield that can be readily adapted for scale-up production. The PEG-SN38 conjugates have shown excellent in vitro anticancer activity, with potency similar to that of native SN38, in a panel of cancer cell lines. The PEG-SN38 conjugates also have demonstrated superior anticancer activity in the MX-1 xenograft mice model compared with CPT-11. Among the four conjugates, PEG-Gly-(20)-SN38 (23) has been selected as the lead candidate for further preclinical development.     

Tripartate poly(ethylene glycol) prodrugs of the open lactone form of camptothecin

Two PEG prodrugs utilizing conjugation of PEG through the C-21 acid functionality as well as the C-17 OH group of CPT hydroxy-amide open forms were synthesized and characterized. Both of these open lactone tripartate prodrugs were shown to be water soluble and highly effective in MX-1 mouse xenograph studies. Indirect evidence implies that the initial ester or carbonate bond breaking is esterase mediated in the first step of the cascade of CPT release.     

Improving the integrity of three-dimensional vascular patterns by poly(ethylene glycol) conjugation

Development of functional tissue-engineering constructs may require that multiple cell types be organized in controlled three-dimensional (3-D) microarchitectures with proper nutrient diffusion and vascularization. In the past few years, a variety of microscale techniques have demonstrated the ability to control protein and cell attachment in defined patterns. Nevertheless, maintenance of these patterns over time has been a significant challenge due to nonspecific protein adsorption and cell migration. To this end, we have investigated the effectiveness of poly(ethylene glycol) (PEG) thin films in maintaining the integrity of 3-D cellular patterns, using human umbilical vein endothelial cells (HUVEC) as a model system. These HUVEC constructs were created using extracellular matrix (ECM)-based microfluidic patterning. Our results indicated that PEG-conjugated substrates improve cell pattern integrity as compared to control silicon. The compliance multifactor (a measure of pattern integrity; higher value means lower pattern integrity) was about 3.66 +/- 0.29 on day 5 for PEG-conjugated surfaces, compared with 8.23 +/- 0.42 for control surfaces ECM-based microfluidic patterning coupled with stable PEG-conjugated surfaces may serve as a vital tool for vascularized tissue engineering.     

Synthesis of multiacyl poly(ethylene glycol) for the conjugation of cytochrome c to phospholipid vesicle

To conjugate water-soluble macromolecules on the surface of phospholipid vesicles, we synthesized a poly(ethylene glycol) (PEG)-lipid having four acyl chains using a lysine (Lys)-type monodendron structure. One end of the diamino-PEG was amidified with Lys, and then two amino groups of the Lys moiety were amidified with two Lys derivatives which had been acylated with two stearoyl groups. The other end of the PEG was activated with a triazine group or a pyridyldithio group. The hydrate of the lipid mixture of dipalmitoylphosphatidylcholine, cholesterol, dipalmitoylphosphatidylglycerol, and the PEG-lipid at a molar ratio of 5/5/1/0.3 was extruded in order to prepare the phospholipid vesicles with the average diameter of 270 +/- 20 nm. The coupling ratio of cytochrome c with the PEG-lipid was monitored by HPLC, detecting the pyridyl 2-thione liberated from the pyridyldithio group and determining it to be 26% on the basis of the incorporated PEG-lipid.     

A facile synthesis of azido-terminated heterobifunctional poly(ethylene glycol)s for "click" conjugation

New azido-terminated heterobifunctional poly(ethylene glycol) (PEG) derivatives having primary amine and carboxyl end groups, (Azide-PEG-NH 2 and Azide-PEG-COOH, respectively) were synthesized with high efficiency. An alpha-allyl-omega-hydroxyl PEG was prepared as the first step to Azide-PEG-X (X = NH 2 and COOH) through the ring-opening polymerization of ethylene oxide (EO) with allyl alcohol as an initiator, followed by two-step modification of the hydroxyl end to an azido group. To introduce primary amino or carboxyl functional groups, amination and carboxylation reactions of the allyl terminal ends was then conducted by a radical addition of thiol compounds. Molecular functionalities of both ends of the PEG derivatives thus prepared were characterized by (1)H, (13)C NMR, and MALDI-TOF MS spectra, validating that the reaction proceeded quantitatively. The terminal azido functionality is available to conjugate various ligands with an alkyne group through the 1,3-dipolar cycloaddition reaction condition ("click chemistry").     

New targeting system for antimycotic drugs: beta-glucosidase sensitive amphotericin B-star poly(ethylene glycol) conjugate

A new targeting potentially intravenous conjugate Amphotericin B (AMB)–star poly(ethylene glycol) (sPEG) (M = 25,160) has been synthesized and characterized. It contains a β-d-glucopyranoside molecular switch which is sensitive to β-glucosidases (E.C.3.2.1.21). The β-glucosidase-catalyzed release of AMB from the polymeric carrier was proved in vitro by means of spectrophotometry and HPLC.     

Synthesis of oligo(poly(ethylene glycol) fumarate)

This protocol describes the synthesis of oligo(poly(ethylene glycol) fumarate) (OPF; 1-35 kDa; a polymer useful for tissue engineering applications) by a one-pot reaction of poly(ethylene glycol) (PEG) and fumaryl chloride. The procedure involves three parts: dichloromethane and PEG are first dried; the reaction step follows, in which fumaryl chloride and triethylamine are added dropwise to a solution of PEG in dichloromethane; and finally, the product solution is filtered to remove by-product salt, and the OPF product is twice crystallized, washed and dried under vacuum. The reaction is affected by the molecular weight of PEG and reactant molar ratio. The OPF product is cross-linked by radical polymerization by either a thermally induced or ultraviolet-induced radical initiator, and the physical properties of the OPF oligomer and resulting cross-linked hydrogel are easily tailored by varying PEG molecular weight. OPF hydrogels are injectable, they polymerize in situ and they undergo biodegradation by hydrolysis of ester bonds. The expected time required to complete this protocol is 6 d.     

Adsorption of poly(ethylene glycol)-modified ribonuclease A to a poly(lactide-co-glycolide) surface

Protein adsorption is a source of variability in the release profiles of therapeutic proteins from biodegradable microspheres. We employ optical reflectometry and total internal reflection fluorescence to explore the extent and kinetics of ribonuclease A (RNase A) adsorption to spin-cast films of poly(lactide-co-glycolide) (PLG) and, in particular, to determine how covalent grafting of polyethylene glycol (PEG) to RNase A affects adsorption. Adsorption kinetics on PLG surfaces are surface-limited for RNase A but transport-limited for unconjugated PEG homopolymers and for PEG-modified RNase A, indicating that PEG anchors the conjugates to the surface during the transport-limited regime. PEG modification of RNase A decreases the total number of adsorbed molecules per unit area but increases the areal surface coverage because the grafted PEG chains exclude additional surface area. Total internal reflection fluorescence-based exchange measurements show that there is no exchange between adsorbed and solution-phase protein molecules. This indicates an unusually tenacious adsorption. Streaming current measurements indicate that the zeta potential of the PLG surface becomes increasingly negative as the film is exposed to water for several weeks, as expected. Aging of the PLG surface results in increased adsorption of unmodified RNase A but decreased adsorption of unconjugated PEG homopolymers and of PEG-RNase A conjugates, relative to the extent of adsorption on freshly prepared PLG surfaces. Adsorption results correlate well with an increase in the rate, total extent and preservation of bioactivity of RNase A released from PLG microspheres for the PEG-modified version of RNase A.     

Enzymatic activation of second-generation dendritic prodrugs: Conjugation of self-immolative dendrimers with poly(ethylene glycol) via click chemistry

Single-triggered disassemble dendrimers were recently developed and introduced as a potential platform for a multi-prodrug. These unique structural dendrimers can release all of their tail units through a self-immolative chain fragmentation initiated by a single cleavage at the dendrimer's core. There are several examples for the bioactivation of first-generation self-immolative dendritic prodrugs. However, enzymatic activation failed for second-generation self-immolative dendrimers. The hydrophobic large molecular structure of the dendritic prodrugs results in aggregation under aqueous conditions and prevented the enzyme from reaching the triggering substrate. Here we show a simple solution for the enzymatic activation of second-generation self-immolative dendrimers. Poly(ethylene glycol) (PEG) was conjugated to the dendritic platform via click chemistry. The poly(ethylene glycol) tails significantly decreased the hydrophobic properties of the dendrimers and thereby prevented aggregate formation. We designed and synthesized a dendritic prodrug with four molecules of the anticancer agent camptothecin and a trigger that can be activated by penicillin-G-amidase. The PEG5000-conjugated, self-immolative dendritic prodrug was effectively activated by penicillin-G-amidase under physiological conditions and free camptothecin was released to the reaction media. Cell-growth inhibition assays demonstrated increased toxicity of the dendritic prodrug upon incubation with the enzyme.     

Mechanism of poly(ethylene glycol) interaction with proteins

Poly(ethylene glycol) (PEG) is one of the most useful protein salting-out agents. In this study, it has been shown that the salting-out effectiveness of PEG can be explained by the large unfavorable free energy of its interaction with proteins. Preferential interaction measurements of beta-lactoglobulin with poly(ethylene glycols) with molecular weights between 200 and 1000 showed preferential hydration of the protein for those with Mr greater than or equal to 400, the degree of hydration increasing with the increase in poly(ethylene glycol) molecular weight. The preferential interaction parameter had a strong cosolvent concentration dependence, with poly(ethylene glycol) 1000 having the sharpest decrease with an increase in concentration. The preferential hydration extrapolated to zero cosolvent concentration increased almost linearly with increasing size of the additive, suggesting steric exclusion as the major factor responsible for the preferential hydration. The poly(ethylene glycol) concentration dependence of the preferential interactions could be explained in terms of the nonideality of poly(ethylene glycol) solutions. All the poly(ethylene glycols) studied, when used at levels of 10-30%, decreased the thermal stability of beta-lactoglobulin, suggesting that caution must be exercised in the use of this additive at extreme conditions such as high temperature.     

Helix stabilization of poly(ethylene glycol)-peptide conjugates

Hybrid polymer-peptide conjugates offer the potential for incorporating biological function into synthetic materials. The secondary structure of short helical peptides, however, frequently becomes less stable when expressed independent of longer protein sequences or covalently linked with a conformationally disordered synthetic polymer. Recently, new amphipathic peptide-poly(ethylene glycol) conjugates were introduced (Shu, J., et al. Biomacromolecules 2008, 9, 2011), which displayed enhanced peptide helicity upon polymer functionalization while retaining tertiary coiled-coil associations. We report here a molecular simulation study of peptide helix stabilization by conjugation with poly(ethylene glycol). The polymer oxygens are shown to favorably interact with the cationic lysine side chains, providing an alternate binding site that protects against disruption of the peptide hydrogen-bonds that stabilize the helical conformation. When the peptide lysine charges are neutralized or poly(ethylene glycol) is conjugated with polyalanine, the polymer exhibits a negligible effect on the secondary structure. We also observe the interactions of poly(ethylene glycol) with the amphipathic peptide lysines tends to segregate the polymer away from the nonpolar face of the helix, suggesting no disruption of the interactions that drive tertiary contacts between helicies.     

Cryopreservation of cell-containing poly(ethylene) glycol hydrogel microarrays

Here we describe the fabrication and preservation of mammalian cell-containing hydrogel microarrays that have potential applications in drug screening and pathogen detection. Hydrogel microstructures containing murine fibroblasts were fabricated on silicon substrates and subjected to a "stage-down" freezing process. The percent viability of both immortal and primary embryonic murine fibroblast cells within the gels was determined at various stages in the freezing process, showing that cells entrapped in hydrogel microstructures remained viable throughout the process. When compared to immortalized adherent cultures subjected to the same freezing process, cells within hydrogel structures had higher cell viabilities at all stages during preservation. Finally, the necessity of using a cryoprotectant, dimethyl sulfoxide (DMSO), was investigated. Cells in hydrogels were cryopreserved with and without DMSO. The addition of DMSO altered cell viability after the freeze-thaw process, enhancing viability in an immortalized cell line and decreasing viability in a primary cell line.     

Analytical partitioning of poly(ethylene glycol)-modified proteins

Covalently grafting proteins with varying numbers (n) of poly(ethylene glycol) molecules (PEGs) often enhances their biomedical and industrial usefulness. Partition between the phases in aqueous polymer two-phase systems can be used to rapidly characterize polymer-protein conjugates in a manner related to various enhancements. The logarithm of the partition coefficient (K) approximates linearity over the range 0<n<x. However, x varies with the nature of the conjugate (e.g., protein molecular mass) and such data analysis does not facilitate the comparison of varied conjugates. The known behavior of surface localized PEGs suggests a better correlation should exist between log K and the weight fraction of polymer in PEG-protein conjugates. Data from four independent studies involving three proteins (granulocyte-macrophage colony stimulation factor, bovine serum albumin and immunoglobulin G) has been found to support this hypothesis. Although somewhat simplistic, ‘weight fraction’ based analysis of partition data appears robust enough to accommodate laboratory to laboratory variation in protein, polymer and phase system type. It also facilitates comparisons between partition data involving disparate polymer-protein conjugates.     

Synthesis and characterisation of a new amphotericin B-methoxypoly(ethylene glycol) conjugate

The reaction of methoxypoly(ethylene glycol)-4-nitrophenyl carbonate with amphotericin B has been used to prepare a new conjugate of amphotericin B (mPEG-AmB). A preliminary screening of in vitro antifungal activity has suggested that mPEG-AmB possesses a similar effect and a similar spectrum of activity as the conventional amphotericin B formulated with sodium desoxycholate.     

UV-patterned poly(ethylene glycol) matrix for microarray applications

A versatile method to fabricate polymeric matrixes for microarray applications is demonstrated. Several different design strategies are presented where a variety of organic films, such as plastic polymers and self-assembled monolayers (SAMs) on planar silica and gold substrates, act as supports for the graft polymerization procedure. An ensemble of poly(ethylene glycol) methacrylate monomers are combined to obtain a matrix with desired properties: low nonspecific binding and easily accessible groups for postimmobilization of ligands. The free radical graft polymerization process occurs under irradiation with UV light in the 254-266 nm range, which offers the possibility to introduce patterns by means of a photomask. The arrays are created on inert and homogeneous coatings prepared either by graft polymerization of a methoxy-terminated PEG-methacrylate or self-assembly of a methoxy-terminated oligo(ethylene glycol) thiol. Carboxylic acid groups, introduced in the array spots either during graft polymerization or upon wet chemical conversion of hydroxyls, grant the capability to immobilize proteins and other molecules via free amine groups. Immobilization of fluorescent species as well as biotin followed by exposure to a fluorescently labeled antibody directed toward biotin display both excellent integrity of the spots and low nonspecific binding to the surrounding framework. Beside patterns of uniform height and size, an array of spots with varying thickness (a sort of gradient) is demonstrated. Such gradient samples enable us to address critical issues regarding the mechanism(s) behind spatially resolved free radical polymerization of methacrylates. It also offers a convenient route to optimize the matrix properties with respect to thickness, loading capacity, protein diffusion/penetration, and nonspecific binding.     

Lipase-catalyzed transformations using poly(ethylene glycol) as solvent

Abstract

Candida antarctica lipase catalyzes a number of elementary reactions like alcoholysis, ammoniolysis and aminolysis in poly(ethylene glycol) (PEG) media. Reaction rates were comparable to or better than those observed in conventional organic reaction media and ionic liquids. It is envisaged that PEGs could have added benefits for performing biotransformations with highly polar substrates, which are sparingly soluble in common organic solvents.     

Probing protein nanopores with poly(ethylene glycol)s

Neutral water-soluble poly(ethylene glycol)s (PEGs) have been extensively explored in protein nanopore research for the past several decades. The principal use of PEGs is to investigate the membrane protein ion channel physical characteristics and transport properties. In addition, protein nanopores are used to study polymer–protein interactions and polymer physicochemical properties. In this review, we focus on the biophysical studies on probing protein ion channels with PEGs, specifically on nanopore sizing by PEG partitioning. We discuss the fluctuation analysis of ion channel currents in response to the PEGs moving within their confined geometries. The advantages, limitations, and recent developments of the approach are also addressed.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M_w=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1±0.8 (mg/m²)/(mg/ml) for 2.5 loops to 78.1±12.2 (mg/m²)/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4±0.1 (mg/m²)/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

Association of hydrophobically-modified poly(ethylene glycol) with fusogenic liposomes

We present results on using cooperative interactions to shield liposomes by incorporating multiple hydrophobic anchoring sites on polyethylene glycol (PEG) polymers. The hydrophobically-modified PEGs (HMPEGs) are comb-graft polymers with strictly alternating monodisperse PEG blocks (M(w)=6, 12, or 35 kDa) bonded to C18 stearylamide hydrophobes. Cooperativity is varied by changing the degree of oligomerization at a constant ratio of PEG to stearylamide. Fusogenic liposomes prepared from N-C12-DOPE:DOPC 7:3 (mol:mol) were equilibrated with HMPEGs. Affinity for polymer association to liposomes increases with the degree of oligomerization; equilibrium constants (given as surface coverage per equilibrium concentration of free polymer) for 6 kDa PEG increased from 6.1+/-0.8 (mg/m(2))/(mg/ml) for 2.5 loops to 78.1+/-12.2 (mg/m(2))/(mg/ml) for 13 loops. In contrast, the equilibrium constant for distearoylphosphatidylethanolamine-poly(ethylene glycol) (DSPE-PEG5k) was 0.4+/-0.1 (mg/m(2))/(mg/ml).The multi-loop HMPEGs demonstrate higher levels of protection from complement binding than DSPE-PEG5k. Greater protection does not correlate with binding strength alone. The best shielding was by HMPEG6k-DP3 (with three 6 kDa PEG loops), suggesting that PEG chains with adequate surface mobility provide optimal protection from complement opsonization. Complement binding at 30 min and 12 h demonstrates that protection by multi-looped PEGs is constant whereas DSPE-PEG5k initially protects but presumably partitions off of the surface at longer times.     

Synthesis and gelation of DOPA-modified poly(ethylene glycol) hydrogels

3,4-Dihydroxyphenylalanine (DOPA) residues are known for their ability to impart adhesive and curing properties to mussel adhesive proteins. In this paper, we report the preparation of linear and branched DOPA-modified poly(ethylene glycol)s (PEG-DOPAs) containing one to four DOPA endgroups. Gel permeation chromatography-multiple-angle laser light scattering analysis of methoxy-PEG-DOPA in the presence of oxidizing reagents (sodium periodate, horseradish peroxidase, and mushroom tyrosinase) revealed the formation of oligomers of methoxy-PEG-DOPA, presumably resulting from oxidative polymerization of DOPA endgroups. In the case of PEG-DOPAs containing two or more DOPA endgroups, oxidative polymerization resulted in polymer network formation and rapid gelation. The amount of time required for gelation of aqueous PEG-DOPA solutions was found to be as little as 1 min and was dependent on the polymer architecture as well as the type and concentration of oxidizing reagent used. Analysis of reaction mixtures by UV-vis spectroscopy allowed the identification of reaction intermediates and the elucidation of reaction pathways. On the basis of the observed reaction intermediates, oxidation of the catechol side chain of DOPA resulted in the formation of highly reactive DOPA-quinone, which further reacted to form cross-linked products via one of several pathways, depending on the presence or absence of N-terminal protecting groups on the PEG-DOPA. N-Boc protected PEG-DOPA cross-linked via phenol coupling and quinone methide tanning pathways, whereas PEG-DOPA containing a free amino group cross-linked via a pathway that resembled melanogenesis. Similar differences were observed for the rate of gel formation as well as the molecular weight between cross-links ((-)M(c)), calculated using equilibrium swelling and the Flory-Rehner equation.