Polymer--drug conjugates as nano-sized medicines

Polymer Therapeutics have enormously evolved in the past decades. Several polymeric drugs as well as polymer-protein conjugates have been in the market since the 90s, but although polymer-drug conjugates are already in clinical trials they still need to reach this final goal. There are four main convergent strategies to move this platform technology further. First, exploitation of new molecular targets in cancer therapy and design of polymer-drug conjugates as treatments for other diseases. Second, the development of combination therapy. Third, attempts to improve polymer chemistry, including the use of new well-defined architectures and the optimization of the advanced characterization techniques essential to transform a promising conjugate into a candidate for clinical evaluation. Finally, increased understanding of polymer conjugate features that govern clinical risk-benefit is leading to an appreciation of clinical biomarkers that will open new possibilities for personalized therapy.     

End-functionalized phosphorylcholine methacrylates and their use in protein conjugation

Polymer-protein conjugation was performed using N-hydroxysuccinimide and aldehyde-terminated zwitterionic polymers, and the resulting polymer-protein conjugates were characterized by gel electrophoresis and fast protein liquid chromatography. Methacryloyloxyethyl phosphorylcholine (MPC) polymers were prepared by atom transfer radical polymerization in which the requisite functional end-groups for protein conjugation were embedded within the polymerization initiators. These phosphorylcholine polymers were conjugated to lysozyme as a model protein, as well as two therapeutic proteins, granulocyte colony stimulating factor (G-CSF) and erythropoietin (EPO). These MPC polymer-protein conjugates represent alternatives to PEGylated proteins, with the potential to provide improved efficacy in a therapeutic treatment relative to the protein itself.     

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.     

Temperature-induced switching of enzyme activity with smart polymer-enzyme conjugates

A method for thermally induced switching of enzyme activity has been developed, based on the site-directed conjugation of end-reactive temperature-responsive polymers to a unique cysteine (Cys) residue positioned near the enzyme active site. The reversible temperature-induced collapse of N,N-dimethylacrylamide (DMA)/N-4-phenylazo-phenylacrylamide (AZAAm) copolymers (DMAAm) has been used as a molecular switch to control the catalytic activity of endoglucanase 12A (EG 12A). The polymer was conjugated to the EG 12A site-directed mutant N55C, directly adjacent to the cellulose binding cleft, and to the S25C mutant, where the conjugation site is more distant. The N55C conjugate displayed a larger activity shutoff efficiency in the collapsed polymer state than the S25C conjugate. Increasing the polymer molecular weight was also shown to increase the shutoff efficiency of the switch. Related to these effects of conjugation site and polymer size, the switching efficiency was found to be strongly dependent on substrate size. With a small substrate, o-nitrophenyl-beta-d-cellobioside (ONPC), there was minimal blocking of enzyme activity when the polymer was in the expanded state. With a large substrate, hydroxyethyl cellulose (HEC), there was a large reduction of enzyme activity in the polymer expanded state, even with relatively small polymer chains, and a further reduction when the polymer was collapsed. Similar general trends for the interactive effects of conjugation site, polymer size, and substrate size were observed for immobilized conjugates. Kinetic studies demonstrated that the switching activity was due to the blocking of substrate association by the collapsed polymers. These investigations provide mechanistic insight that can be utilized to design molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

多糖-药物轭合物的研究与展望

多糖类物质作为赋形剂在药物制剂中已被广泛使用,多糖结构中包含了多种活性基团如羟基、羧基、氨基等,具有良好的亲水性、生物可降解性以及生物安全性,使其在聚合物-药物轭合物的构建中成为理想的载体材料.目前天然的多糖大分子及其衍生物作为药物载体的研究方兴未艾,以多糖为载体的聚合物-药物轭合物在定位或靶向给药、组织工程、生物黏附等领域也备受关注.本文以天然多糖-药物轭合物的研究现状为切入点,总结归纳了多糖-药物轭合物的设计与构建途径,介绍了其在药物传递中的应用,讨论并分析了多糖在轭合物体系中的角色和发挥的作用,对以多糖为载体的聚合物-药物轭合物发展的方向予以了讨论.     

Pentafluorophenyl ester-functionalized phosphorylcholine polymers: preparation of linear, two-arm, and grafted polymer-protein conjugates

Novel pentafluorophenyl (PFP)-ester-functionalized phosphorylcholine (PC) polymers of different architectures were prepared and conjugated to lysozyme as a model protein. Linear and two-arm poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) structures containing PFP functionality at the chain-end were prepared by atom transfer radical polymerization (ATRP) from novel initiators. Additional conjugates were prepared from phosphorylcholine-substituted cyclooctene (PC-COE) polymers containing PFP-ester bearing comonomers. The polymer-protein conjugates were characterized by HPLC, FPLC, and DLS and were seen to retain most (~80% or greater) of their native enzymatic activity. Pharmacokinetic profiles of the polymer-protein conjugates were studied in mice and found to increase the circulation half-life compared with lysozyme alone.     

"Starburst" conjugates of proteins with carbocene polymers,carrying low molecular weight biologically-active compounds,and their immunogenicity

The synthesis of starburst polymer-protein conjugates on the basis of bovine serum albumin and horseradish peroxidase was performed with the aim to study the possibilities of regulation of the immune response against the components of the conjugates. These polymers had one-point binding between the protein and the modifying carbon-chain polymer that contained 1-vinyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline (a salsolinol analogue) or bradykinin (peptide hormone) residues in its carbon chain. Changes in the chemical nature of the carbon-chain part of the polymer-protein conjugate were shown to increase or decrease the level of antibody production both against the low-molecular compounds attached to the polymeric fragments and against the protein carrier.     

Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

Synthesis, solution conformation, and antibody recognition of oligotuftsin-based conjugates containing a beta-amyloid(4-10) plaque-specific epitope

One possible therapeutic approach to treat or prevent Alzheimer's disease (AD) is immunotherapy. On the basis of the identification of Abeta(4-10) (FRHDSGY) as the predominant B-cell epitope recognized by therapeutically active antisera from transgenic AD mice, conjugates with defined structures containing the epitope peptide attached to a tetratuftsin derivative as an oligopeptide carrier were synthesized and their structure characterized. To produce immunogenic constructs, the Abeta(4-10) epitope alone or flanked by alpha- or beta-alanine residues was attached through an amide bond to the tetratuftsin derivative (Ac-[TKPKG]4-NH2) or to a carrier peptide elongated by a promiscuous T-helper cell epitope (Ac-FFLLTRILTIPQSLD-[TKPKG]4-NH2). The conformational preferences of the carrier and conjugates were examined by CD spectroscopy in water and in 1:1 and 9:1 TFE:water mixtures (v/v). We found that the presence of flanking dimers in the conjugates had no effects on the generally unordered solution conformation of the conjugates. However, conjugates with an elongated peptide backbone exhibited CD spectra indicative for a partially ordered secondary structure in the presence of TFE. Comparative ELISA binding studies, using monoclonal antibody raised against the beta-amyloid (1-17) peptide, showed that conjugates with T-helper cell epitope in the carrier backbone exhibited decreased monoclonal antibody recognition. However, we found that this effect was compensated in conjugates comprising the Abeta(4-10) B-cell epitope with the beta-alanine dimer flanking regions at both N- and C-termini. Results suggest that modification of the B-cell epitope peptide from Abeta with rational combination of structural elements (e.g. conjugation to carrier, introduction of flanking dimers) can result in synthetic antigen with preserved antibody recognition.     

Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.     

Mechanistic investigation of smart polymer-protein conjugates

Many affinity separation and diagnostic applications rely upon both capture and release steps. There is thus a need for methods to enhance the reversibility of biomolecular interactions. We have previously demonstrated that stimuli-responsive polymers can be used to gate biomolecular reactions when conjugated near the active site of proteins. Here we have used a new smart polymer, N,N-dimethyl acrylamide-co-4-phenylazophenylacrylate that has allowed a mechanistic investigation of the smart polymer switches. This polymer was conjugated via a vinyl sulfone terminus to cysteine residues of genetically engineered streptavidin mutant E116C, where the polymer is conjugated close to the biotin-binding site, and streptavidin mutant S139C, where the conjugation site is distant. The biotin binding switching activity was strongly dependent on conjugation position, as the E116C conjugate displayed a large thermal response while the S139C conjugate displayed only small effects. Kinetic measurements of biotin release demonstrated that the off-rate of biotin was unperturbed and that the thermally triggered release of biotin with the E116C conjugate was due to the blocking the reassociation of biotin. The addition of free polymer to purified E116C conjugates was also shown to increase the blocking and release properties of the switch. This effect was site dependent, suggesting that the conjugated polymers were directing a physical aggregation near the binding site that effectively enhanced the switching activity. These investigations provide mechanistic insight that can be utilized to design better molecular switches for a variety of stimuli-responsive polymer-protein conjugates.     

Reagents for astatination of biomolecules. 5. Evaluation of hydrazone linkers in (211)At- and (125)I-labeled closo-decaborate(2-) conjugates of Fab' as a means of decreasing kidney retention

Evaluation of monoclonal antibody (mAb) fragments (e.g., Fab', Fab, or engineered fragments) as cancer-targeting reagents for therapy with the α-particle emitting radionuclide astatine-211 ((211)At) has been hampered by low in vivo stability of the label and a propensity of these proteins localize to kidneys. Fortunately, our group has shown that the low stability of the (211)At label, generally a meta- or para-[(211)At]astatobenzoyl conjugate, on mAb Fab' fragments can be dramatically improved by the use of closo-decaborate(2-) conjugates. However, the higher stability of radiolabeled mAb Fab' conjugates appears to result in retention of radioactivity in the kidneys. This investigation was conducted to evaluate whether the retention of radioactivity in kidney might be decreased by the use of an acid-cleavable hydrazone between the Fab' and the radiolabeled closo-decaborate(2-) moiety. Five conjugation reagents containing sulfhydryl-reactive maleimide groups, a hydrazone functionality, and a closo-decaborate(2-) moiety were prepared. In four of the five conjugation reagents, a discrete poly(ethylene glycol) (PEG) linker was used, and one substituent adjacent to the hydrazone was varied (phenyl, benzoate, anisole, or methyl) to provide varying acid sensitivity. In the initial studies, the five maleimido-closo-decaborate(2-) conjugation reagents were radioiodinated ((125)I or (131)I), then conjugated with an anti-PSMA Fab' (107-1A4 Fab'). Biodistributions of the five radioiodinated Fab' conjugates were obtained in nude mice at 1, 4, and 24 h post injection (pi). In contrast to closo-decaborate(2-) conjugated to 107-1A4 Fab' through a noncleavable linker, two conjugates containing either a benzoate or a methyl substituent on the hydrazone functionality displayed clearance rates from kidney, liver, and spleen that were similar to those obtained with directly radioiodinated Fab' (i.e., no conjugate). The maleimido-closo-decaborate(2-) conjugation reagent containing a benzoate substituent on the hydrazone was chosen for study with (211)At. That reagent was conjugated with 107-1A4 Fab', then labeled (separately) with (125)I and (211)At. The radiolabeled Fab' conjugates were coinjected into nude mice bearing LNCaP human tumor xenografts, and biodistribution data were obtained at 1, 4, and 24 h pi. Tumor targeting was achieved with both (125)I- and (211)At-labeled Fab', but the (211)At-labeled Fab' reached a higher concentration (25.56 ± 11.20 vs 11.97 ± 1.31%ID/g). Surprisingly, while the (125)I-labeled Fab' was cleared from kidney similar to earlier studies, the (211)At-labeled Fab'was not (i.e., kidney conc. for (125)I vs (211)At; 4 h, 13.14 ± 2.03 ID/g vs 42.28 ± 16.38%D/g; 24 h, 4.23 ± 1.57 ID/g vs 39.52 ± 15.87%ID/g). Since the Fab' conjugate is identical in both cases except for the radionuclide, it seems likely that the difference in tissue clearance seen is due to an effect that (211)At has on either the hydrazone cleavage or on the retention of a metabolite. Results from other studies in our laboratory suggest that the latter case is most likely. The hydrazone linkers tested do not provide the tissue clearance sought for (211)At, so additional hydrazones linkers will be evaluated. However, the results support the use of hydrazone linkers when Fab' conjugated with closo-decaborate(2-) reagents are radioiodinated.     

Separation of PEGylated alpha-lactalbumin from unreacted precursors and byproducts using ultrafiltration

There is considerable clinical interest in the use of "second-generation" therapeutic proteins produced by conjugation of the native protein with various polymers including poly(ethylene glycol) (PEG). One of the challenges in the production of polymer-protein conjugates is the need to remove residual polymer, native (unreacted) protein, and any reaction byproducts from the final therapeutic formulation. The overall objective of this study was to evaluate the possibility of using ultrafiltration for the purification of a model PEGylated protein. Sieving data were obtained using PEGylated alpha-lactalbumin, the native protein, and the poly(ethylene glycol) over a range of pH, ionic strength, and filtrate flux using both neutral and charge-modified composite regenerated cellulose membranes. Purification of the PEGylated protein was achieved using a two-stage diafiltration process. The first stage used a neutral membrane to remove the unreacted protein and any small reaction byproducts while retaining the large PEGylated product. The second stage used a negatively charged membrane to remove the neutral poly(ethylene glycol) while retaining the PEGylated alpha-lactalbumin as a result of strong electrostatic interactions. These results clearly demonstrate the potential of using membrane-based separations for the purification of second-generation therapeutic proteins.     

Squaric acid mediated synthesis and biological activity of a library of linear and hyperbranched poly(glycerol)-protein conjugates

Polymer-protein conjugates generated from side chain functional synthetic polymers are attractive because they can be easily further modified with, for example, labeling groups or targeting ligands. The residue specific modification of proteins with side chain functional synthetic polymers using the traditional coupling strategies may be compromised due to the nonorthogonality of the side-chain and chain-end functional groups of the synthetic polymer, which may lead to side reactions. This study explores the feasibility of the squaric acid diethyl ester mediated coupling as an amine selective, hydroxyl tolerant, and hydrolysis insensitive route for the preparation of side-chain functional, hydroxyl-containing, polymer-protein conjugates. The hydroxyl side chain functional polymers selected for this study are a library of amine end-functional, linear, midfunctional, hyperbranched, and linear-block-hyperbranched polyglycerol (PG) copolymers. These synthetic polymers have been used to prepare a diverse library of BSA and lysozyme polymer conjugates. In addition to exploring the scope and limitations of the squaric acid diethylester-mediated coupling strategy, the use of the library of polyglycerol copolymers also allows to systematically study the influence of molecular weight and architecture of the synthetic polymer on the biological activity of the protein. Comparison of the activity of PG-lysozyme conjugates generated from relatively low molecular weight PG copolymers did not reveal any obvious structure-activity relationships. Evaluation of the activity of conjugates composed of PG copolymers with molecular weights of 10000 or 20000 g/mol, however, indicated significantly higher activities of conjugates prepared from midfunctional synthetic polymers as compared to linear polymers of similar molecular weight.     

Impact of aldehyde content on amphotericin B-dextran imine conjugate toxicity

The biocompatibility of oxidized dextran (40 kDa) was investigated in vitro. The contribution of aldehyde groups to the toxicity of polymer-drug conjugates, such as dextran-amphotericin B (AmB) was evaluated. Oxidized dextran was proved to be toxic against the RAW 264.7 cell line with an IC50 of 3 micromol/mL aldehydes. Modification of aldehyde groups and their reaction with ethanolamine reduced the toxicity at least 15-fold. Accordingly, the antifungal and antileishmanial dextran-AmB imine conjugate, which contains unreacted aldehyde groups, was modified with ethanolamine and compared to dextran-AmB amine and imine conjugates. Modification of the imine conjugate with ethanolamine reduced its toxicity toward the RAW cell line by 100%. The effect on Leishmania major parasites was 5 times higher than that of the dextran-AmB amine conjugate. The dextran-AmB-ethanolamine conjugate was at least 15 times less hemolytic than free AmB. Stability and drug release profiles in buffer solution were investigated. The imine conjugates released free AmB while the amine conjugate did not. It is concluded that aldehyde groups may contribute to cell toxicity. This toxicity is reduced by converting the aldehyde groups into imine conjugates with ethanolamine. The results have direct implications toward the safety of AmB-polysaccharide conjugates used against fungal and leishmanial infections.     

Anionic surfactants enhance click reaction-mediated protein conjugation with ubiquitin

The Cu(I)-catalyzed alkyne–azide cycloaddition (CuAAC) has become increasingly important in the conjugation chemistry of biomolecules. For example, it is an efficient and convenient method to generate defined ubiquitin–protein conjugates. Here, we investigate the effect of surfactants on the efficiency of CuAAC for chemical protein ubiquitylation. We found that anionic surfactants enhance conjugate formation by up to 10-fold resulting in high yields even at low (i.e., micromolar) concentrations of the reactants. Notably, the herein investigated conjugates are functional and thus properly folded.     

Immobilization and intracellular delivery of an anticancer drug using mussel-inspired polydopamine capsules

We report a facile approach to immobilize pH-cleavable polymer-drug conjugates in mussel-inspired polydopamine (PDA) capsules for intracellular drug delivery. Our design takes advantage of the facile PDA coating to form capsules, the chemical reactivity of PDA films, and the acid-labile groups in polymer side chains for sustained pH-induced drug release. The anticancer drug doxorubicin (Dox) was conjugated to thiolated poly(methacrylic acid) (PMA(SH)) with a pH-cleavable hydrazone bond, and then immobilized in PDA capsules via robust thiol-catechol reactions between the polymer-drug conjugate and capsule walls. The loaded Dox showed limited release at physiological pH but significant release (over 85%) at endosomal/lysosomal pH. Cell viability assays showed that Dox-loaded PDA capsules enhanced the efficacy of eradicating HeLa cancer cells compared with free drug under the same assay conditions. The reported method provides a new platform for the application of stimuli-responsive PDA capsules as drug delivery systems.     

Vinyl sulfone bifunctional derivatives of DOTA allow sulfhydryl- or amino-directed coupling to antibodies. Conjugates retain immunoreactivity and have similar biodistributions.

We have synthesized a bifunctional vinyl sulfone-cysteineamido derivative of DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) that can be conjugated to the sulfhydryls of mildly reduced recombinant antibody (chimeric anti-CEA antibody cT84.66) at pH 7 or to the amino groups of lysine residues at pH 9. The conjugation is sulfhydryl specific at pH 7 (case 1), and amino specific at pH 9 (case 2) as long as the antibody has no free sulhydryl groups. At a molar ratio of 50 BCA (bifunctional chelating agent) to mAb, the number of chelates conjugated is 0.8 for case 1, and 4.6 for case 2. The resulting conjugates can be radiolabeled with (111)In to high specific activity (5 mCi/mg) with high efficiency (>95%) at 43 degrees C in 60 min. The radiolabeled conjugates retained >95% immunoreactivity and are stable in serum containing 1mM DTPA over 3 d. When the radiolabeled conjugates were injected into nude mice bearing LS174T human colon tumor xenografts, over 40% ID/g accumulated in tumors during the period 24-72h. Tumor-to-blood ratios were 4.5, 3.5, and 2.5 for the sulfhydryl coupled conjugate at 24, 48, and 72 h, respectively, and 2.7, 2.5, and 2.3 for the amino-coupled conjugate at the same time points. For other organs the biodistributions were nearly identical whether the conjugates were attached via sulfhydryl or amino groups. These novel BCAs are easy to synthesize, offer versatile conjugation options, and give equivalent biodistributions that result in high tumor uptake and good tumor-to-blood ratios.     

Drug delivery systems: polymers and drugs monitored by capillary electromigration methods

In this paper, different electromigration methods used to monitor drugs and polymers released from drug delivery systems are reviewed. First, an introduction to the most typical arrangements used as drug delivery systems (e.g., polymer-drug covalent conjugates, membrane or matrix-based devices) is presented. Next, the principles of different capillary electromigration procedures are discussed, followed by a revision on the different procedures employed to monitor the release of drugs and the degradation or solubilization of the polymeric matrices from drug delivery systems during both in vitro and in vivo assays. A critical comparison between these capillary electrophoretic methods and the more common chromatographic methods employed to analyze drugs and polymers from drug delivery systems is presented. Finally, future outlooks of these electromigration procedures in the controlled release field are discussed.