Semisynthetic Hydrophilic Polyals - Biomacromolecules (ACS

Biomacromolecules 2005, 6, 2659-2670

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Semisynthetic Hydrophilic Polyals Mikhail I. Papisov,* Alexander Hiller, Alexander Yurkovetskiy, Mao Yin, Marlene Barzana, Shawn Hillier, and Alan J. Fischman Department of Radiology, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114-2696 Received March 22, 2005; Revised Manuscript Received June 22, 2005

Nonbioadhesive, fully biodegradable soluble polymers would be very instrumental in advanced biomedical applications, such as gene and drug delivery and tissue engineering. However, rational development of such materials is hindered by the complexity of macromolecule interactions with biological milieu. The prevalence of carbohydrates in naturally occurring interface structures suggests an alternative, biomimetic approach. Interface carbohydrates, regardless of their biological function, have common nonsignaling substructures (e.g., acetal and ketal groups, secondary and primary alcohols). We hypothesized that hydrophilic polymers (polyals) consisting of acyclic units built of nonsignaling carbohydrate substructures would be highly biocompatible and nonbioadhesive, while intrachain acetal or ketal groups would enable nonenzymatic biodegradation upon uptake by cells. Acyclic hydrophilic polyals can be prepared via either polymerization of suitable monomers or lateral cleavage of cyclic polyals (e.g., polysaccharides). In this study, model polyals were produced via lateral cleavage of polyaldoses and polyketoses. Best results were achieved using dextran B-512 as a precursor. The resultant poly[hydroxymethylethylene hydroxymethylformal], in agreement with the hypothesis, demonstrated excellent biological properties and technological flexibility. Materials of this type can potentially have several applications in pharmacology and bioengineering. Introduction Soluble polymers are widely used in pharmacology (currently, primarily as pharmaceutical excipients). They are expected to find several new biomedical applications as structural and interface components of functional macro- and supramolecular systems that are being developed for drug delivery, gene therapy, tissue engineering, and other advanced biomedical applications. Novel concepts of pharmacology and bioengineering impose new, more specific and more stringent requirements on macromolecular components. Ideally, biomedical polymers would be technologically adaptable, completely biodegradable, nontoxic, and cause no adverse reactions of any type. Polymer structure should support an ample set of technologies, such as conjugation with drug molecules, target-specific ligands, and other molecular modules. This translates into the problem of developing macromolecules that have minimal interactions with any cells and biomolecules, completely biodegradable main chains, nontoxic degradation products, and readily modifiable functional groups. None of the currently available materials meet all of the above requirements. For example, poly(ethylene glycol) and other “stealth” polymers have limited biodegradability. Many other synthetic polymers release toxic products upon degradation. Proteins and polysaccharides, on the other hand, are not entirely biologically inert (i.e., not free from * To whom correspondence should be addressed. Phone: (617) 7249655. Fax: (617) 724-8315. E-mail: [email protected].

interactions with cell receptors, recognition proteins, and other components of biological milieu). The biologically important interactions leading to macromolecule internalization by cells, cell adhesion to polymercoated surfaces, and anaphylactoid reactions can be mediated by several cell surface elements, most of which are functionally specialized (receptors, adhesion molecules, etc.). Such interactions are often mediated by specialized recognition proteins of plasma, such as immunoglobulins, fibronectins, proteins of complement system, soluble lectins, etc. The distinctive features of recognition proteins, which bind to a variety of structures, relate to their ability to trigger remarkable biological responses. We reviewed their role in pharmacology of macromolecules and particles in more detail elsewhere.1,2 To date, there is enough knowledge on the specificities of major receptors and recognition proteins to enable selection of materials that are free of structures capable of strong specific binding in vivo. However, the possibility of weak binding still cannot be reliably prevented. Another major factor of macromolecule (and surface) reactivity in vivo is cooperative (multipoint) binding, which is often referred to as “nonspecific interactions”. Strong nonspecific binding is usually caused by electrostatic and hydrophobic interactions. The latter can be minimized, for example, via using only nonionic hydrophilic structures. However, because the cooperative binding energy is additive, the association constant of cooperative binding (Ka) would grow with the number of weak associations exponentially. Therefore, even very weak associations may be cooperatively amplified to a significant level.1 Therefore, in view of the

10.1021/bm0502157 CCC: $30.25 © 2005 American Chemical Society Published on Web 08/18/2005

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Figure 1. Typical structure of an oligosaccharide interface fragment of a glycolipid (shown: GM1). Structures responsible for carbohydrate ring recognition are shown as thin lines; the ubiquitous nonsignaling polyacetal substructure is shown as bold lines.

great diversity of interface structures in a mammalian organism, any polymer of a sufficient length can be expected to interact with at least one type of the various available binding sites. If a macromolecule of a certain type and size is nonbioadhesive, a larger molecule or an assembly of molecules of the same type can be strongly bioadhesive (including selective interactions with a certain cell type3). The above generally suggests that any large macromolecular construct, even assembled of apparently “inert” domains, can still have significant cooperative interactions with at least some tissue component(s). In other words, a completely “bio-inert” polymer may not exist at all. However, several biomolecules and biological interfaces do appear to be essentially inert. We hypothesized that the mutual “inertness” of the biological interfaces may relate to the ubiquity of carbohydratemodified interface structures, where the potential “nonspecific” binding sites are always saturated with naturally occurring counteragents (e.g., glycoproteins).2 Accordingly, emulation of the most ubiquitous interface substructures (excluding their signaling domains) can result in materials that would not actively interact with the existing binding sites. Oligosaccharides are omnipresent on cell surfaces, plasma proteins, and proteins of extracellular matrix and thus appear to be the best candidates for structural emulation. Regardless of their biological function, all interface carbohydrates have common structural fragments (acetal or ketal groups, primary and secondary alcohols). The receptor specificity of each carbohydrate depends on the special configuration of the rigid sets of OH (or other) groups positioned on the carbohydrate ring (Figure 1). The existence of long-circulating (therefore nonbioadhesive and nonsignaling) sialoglycoconjugates suggests that flexible acyclic structures bearing primary and secondary OH groups are generally nonbioadhesive and nonsignaling. Thus, a polymer comprising a flexible polyacetal or polyketal backbone, and primary or secondary OH groups as substituents, can be expected to be nonbioadhesive and biologically inert. If the acetal/ketal oxygens are positioned within the main chain, the polymer will be pHsensitive (hydrolytic depolymerization in acidic conditions).

Materials of the suggested general structure (acyclic hydrophilic polyals) can be produced via a variety of synthetic methods. Alternatively, continuous acyclic polyal chains can be “cleaved out” of some polysaccharides. This paper describes the preparation, isolation, and derivatization of model acyclic hydrophilic polyals from polysaccharides and discusses the basic properties of these polymers and their model derivatives in the context of their potential biomedical applications. Materials and Methods Materials and Equipment. Samples of dextran B-512 (Leuconostoc Mesenteroides) of various molecular weights were from Sigma, St. Louis, MO, Polysciences, Warrington, PA, and other sources. Other polysaccharides were from Sigma: “galactan” from gum arabic (cat.# 851396); mannan from Saccharomyces cereVisae (M7504); levan from Erwinia herbicola (L8647); amylose from potato (A0512); and inulin from chicory root (I2255). Radionuclides were from Perkin-Elmer Life Sciences, MA (formerly NEN Life Sciences). Solvents and other reagents, including sodium m-periodate (ACS reagent grade), polyL-lysine hydrobromide, N-ethyl-N′-(N-diethylaminopropyl)carbodiimide (EDC), and other reagents, were from Sigma. Polymer isolation and fractionation on the subgram scale were carried out by size exclusion chromatography (SEC) on Sephadex G-25 (desalting) or Sephacryl S-100 (fractionation). Large-scale separations were performed using a flow dialysis system (CH2PR, Amicon; presently Millipore, MA) equipped with hollow fiber cartridges with 3 kDa to 1 µm cutoff. Size exclusion HPLC (HPSEC) was carried out using Rainin Dynamax and Varian-Prostar HPLC systems equipped with a BIO-RAD model 1755 Refractive Index detector, LDC/Milton Roy SpectoMonitor 3000 UV detector, and a custom high sensitivity gamma radiation detector. Size exclusion HPLC was performed using BioRad BioSil-125 and other BioSil columns calibrated with standard BioRad protein kit and commercial dextran and PEG size standards. The molecular weights of all polymers, except nominal

Semisynthetic Hydrophilic Polyals

molecular weights of commercial dextran B-512 preparations, are given as effective values calculated from HPSEC. All reactions were carried out in magnetically stirred glass reactors. Deionized water (14-18 MΩ/cm) was used in all experiments. Ambient temperature was 23 ( 3 °C. All animals (20-30 g male CD-1 mice, 150-350 g Sprague-Dawley CD rats, 1.5 kg white NZ rabbits) were from Charles River Laboratories, MA. Scintigraphy was performed using a gamma camera (Ohio Nuclear) equipped with a medium energy collimator and a Technicare 560 image processing system. Synthesis of Acyclic Polyals via Exhaustive Periodate Oxidation of Polysaccharides. The main synthetic objective of this work was to optimize the periodate reaction for complete ring cleavage in polysaccharides with minimal depolymerization. Periodate oxidation in aqueous media is known to be highly specific to 1,2-glycols. Products of partial polysaccharide oxidation (e.g., “dialdehyde dextran”) were used in several bioconjugates. This reaction (“Smith Degradation” variant) was also used for studying carbohydrate structure.4 The latter method has not been intended for aggressive ring cleavage; in fact, oxidation was carried out such that a substituted ring (e.g., C3-substituted pyranoside) would remain fully intact. Isolation of macromolecular products was not an objective of the analytical Smith procedure: products of oxidation were reduced with borohydride and hydrolyzed without isolation for the subsequent fragment analysis. In unsubstituted pyranosides, such as poly-(1f6)-R-Dglucose, oxidation reportedly starts from breaking either the C2-C3 or the C3-C4 bond5 with formation of dialdehyde IIa or IIb (Figure 2). The content of the intermediate incompletely oxidized structures (e.g., IIa vs IIb) is reportedly determined by kinetic factors and depends on the carbohydrate ring structure. Further oxidation, with C3 cleavage (in the form of formic acid), which is significantly slower, results in a dialdehyde III. The latter can be subsequently reduced, for example, with sodium borohydride in aqueous media, with formation of alcohol pendant groups IV. The intermediate incompletely cleaved acyclic polymers (IIa/IIb) can also be reduced, with formation of glycol pendant groups (V). The length (molecular weight) of the oxidation products can depend on the polysaccharide structure; irregularities can result in chain disintegration; such variations in the structure, depending on the source, can always be anticipated. Substituted glycols (branching points) and some other carbohydrate derivatives also react with periodate, but the reaction rates are usually lower. Most data in this report were obtained with dextran B-512. Dextrans are polyglucopyranosides; dextran B-512 is one of the most extensively studied 1f6 polysaccharides.6 According to the literature, dextran B-512, as well as essentially the same dextran B-512 F, is a nearly linear (1f6)-poly-RD-glucose with a relatively small (5-7%) number of short branches connected to the main chain via (1f3; β) bonds.7 Although this structure appeared to be suitable for conversion into high molecular weight acyclic polyacetals, our first attempts to isolate macromolecular oxidation products (utilizing the conventionally used techniques) resulted either in

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Figure 2. Periodate oxidation of poly-1f6 pyranoside (I). Initially, oxidation results in the cleavage of either the C3-C4 or the C2-C3 bond with formation of acyclic structures IIa and IIb. Carbon 3 is then eliminated with formation of carbonylpolyal III (PCF). Borohydride reduction of II and III leads to IV and V, respectively. PHF (IV) is a copolyacetal of glycerol (A) and glycol aldehyde (B).

the preservation of several uncleaved rings or in significant depolymerization. Depolymerization was most significant (a) during the oxidation, and (b) immediately after the addition of borohydride to the oxidation product. The reaction conditions and the order of reagent addition were both optimized to achieve an essentially complete ring cleavage and prevent depolymerization. The below optimized technique enabled conversion of several polysaccharides into acyclic polyacetals. Optimized Procedure for Polyal Synthesis (Representative Example). (A) Oxidation of Polysaccharides. Dextran B-512, 178 kDa, 100 g, was dissolved in 0.5 L of water under vigorous stirring at ambient temperature. Sodium m-periodate, 280 g, was dissolved in 2 L of water. The latter solution was filtered and transferred into a 3 L stirred lightprotected reactor. Both solutions were then cooled to 15 °C (ice bath), and the dextran solution was slowly added under vigorous stirring to the periodate solution, maintaining t < 20 °C (the reaction is exothermic). The ice bath was then removed, and the reaction mixture was incubated at ambient temperature for 24 h under stirring. After the incubation, the reaction mixture was filtered through a 1 µm cutoff hollow fiber cartridge (flow dialysis system, filtration mode). The filtrate was concentrated on a 30 kDa cartridge to 1000 mL. Subsequently, 6 L of deionized water was passed through the concentrate (flow dialysis mode). The resultant carbonyl-polyal solutions were stored at 4 °C (slow reversible association with gel formation was

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observed at pH 5-7) or used immediately. Aliquots were lyophilized to determine polymer content in the solution and to study the properties of the macromolecular carbonylpolyaldehyde III (poly-[carbonylethylene carbonylformal], or PCF). In syntheses on a smaller scale, as well as syntheses based on low molecular weight precursors (MW < 20 kDa), the products were purified via size exclusion chromatography instead of flow dialysis. Sodium periodate excess was inactivated with glycerol, and the macromolecular products were separated from the reaction mixture by gel chromatography on Sephadex G-25. (B) Reduction of Carbonyl Polyals. A calculated amount of carbonyl-polyal III was slowly added to a 20% excess of 10% sodium borohydride under vigorous stirring at 1015 °C. The reaction mixture was incubated under stirring at ambient temperature for 2-8 h and concentrated (flow dialysis system) as much as the viscosity allowed, usually to a polymer content of ca. 10-20%. The concentrate was neutralized (pH ) 6.5-7) with 5 N HCl at 0 °C under vigorous stirring. When hydrogen release stopped, 6 volumes of deionized water was passed through the reaction mixture (flow dialysis) to remove salts. The resultant solution was filtered (1 µm cartridge) and lyophilized to obtain dry polymer IV (poly-[hydroxymethylethylene hydroxymethylformal], or PHF). In smaller scale and lower molecular weight syntheses, the polyals were neutralized and isolated by gel chromatography on Sephadex G-25 instead of flow dialysis. The technique was found to be feasible for producing acyclic polyals from several other polysaccharides (both polyaldoses and polyketoses), although generally with lower yields and more significant depolymerization than from dextran B-512F. All products were obtained in the form of colorless aqueous solutions and (lyophilized) solids. Characterization. All products were studied by HPSEC to determine the extent of depolymerization with respect to the precursor polysaccharides. Polyals obtained with sufficiently high yields were studied by 1H NMR ([D6]DMSO or D2O) and 13C NMR (water, D2O). These included the products of from levan from Erwinia Herbicola (2f6-poly-β-fructose), inulin (2f1-polyβ-fructose), “galactan” from gum Arabic (a highly branched 1f3 linked polygalactose, with 1f6 linked ramified side chains containing arabinose, rhamnose, and glucuronic acid8), and mannan from Saccharomyces cereVisae (1f6 linked mannan backbone with 1f3 linked, 1f2 attached branches9). Products of full dextran B-512 cleavage (III, IV) as well as partial cleavage (II and V) of various molecular weights were further characterized in more detail. Acyclic Polyacetals Generated from Dextran B-512. Solubility was tested using lyophilized samples (50 ( 5 mg) of various molecular weights in glass test tubes; solvents (200 µL) were added at 25 °C. The test tubes were sealed and incubated at 25 °C for 10 h, with periodical stirring (Vortex). Molecular size distributions were determined by HPSEC in 10 mM NaCl. Molecular weights were estimated on the

Papisov et al.

basis of the random coil model, using linear (dextran) and globular (proteins) HPSEC standards. Lyophilized macromolecular products were redissolved in [d6]DMSO or D2O for 1H NMR studies, and in D2O for 13C NMR studies. NMR spectroscopy studies were performed at 293 K, in 10% solutions on a 9.4 T Brucker system (100.619 MHz by 13C, 400.13 MHz by 1H). Polymer hydrolysis was investigated in buffer-free media, using a high molecular weight fraction of PHF (80% within 160 ( 20 kDa by HPLC). The latter was obtained via partial SEC fractionation of a broader molecular weight sample. PHF was dissolved in water (1 mg/mL), and the pH was adjusted with either HCl or NaOH to cover the range from 2 to 12. The solutions were then incubated for 11 days at 37 °C in sealed test tubes. Periodically, samples were taken for HPSEC, and the pH was adjusted when necessary. To determine whether PHF retained any structures recognizable by dextran-specific antibodies, dextran B-512 (178 kDa) and PHF obtained by lateral cleavage of the same dextran were dissolved in isotonic (NaCl) 50 mM PBS, pH ) 7.5. Dextran-specific antibodies (DX-1, StemCell technologies, USA) were added at 1:20 w/w to either polymer. After a 1 h incubation at ambient temperature, the reaction mixtures were studied by HPSEC with UV and refraction index detection. Derivatives. Acylation. PHF was prepared, as described above, using 180 kDa dextran B-512. The synthesized polymer had a broad molecular weight distribution similar to the original polysaccharide, with peak MW of ca. 100 kDa. Diethylenetriaminepentaacetic acid (DTPA) anhydride (1 mg) was added to a solution of 100 mg of PHF and 5 µL of triethylamine in 0.5 mL of DMSO; the reaction mixture was stirred for 30 min at 50 °C and then for 24 h at 25 °C, diluted 3:1 with water, and the DTPA-modified polymer was purified by gel chromatography on Sephadex G25/water. The polymer was labeled with 111In by transchelation from carrierfree [111In] indium citrate in 0.5 M citrate buffer solution, pH ) 5.6, desalted on Sephadex G25 with buffer exchange to isotonic (0.9%) NaCl, and analyzed by HPSEC. Alkylation (Cross-Linked Gels). PHF was prepared as described above from technical grade 2 MDa dextran B-512 and fractionated by ultrafiltration to obtain a fraction with 90% MW within the 100-500 kDa range. The solution of 760 mg of this polymer in 5 mL of water was mixed with 5 mL of 10 N NaOH. The resultant solution was distributed into four 16 mm test tubes, 2 mL in each. Epibromohydrine was added to each test tube: 20 µL (#1), 50 µL (#2), 100 µL (#3), and 200 µL (#4). The mixtures were vigorously stirred at ambient temperature for 1 min to emulsify epybromohydrine, then incubated at 80 °C for 8 h to form cross-linked gels (Scheme 1). Cross-linking of PHF molecules, as in the above scheme, results in the formation of gels. Cross-link density is defined by the amount of epybromohydrine introduced into the reaction. After the incubation, gels were removed from the test tubes, cut into 0.5 cm thick disks, and washed in a continuous flow of deionized water overnight. The obtained gel blocks were incubated in aqueous media, with registration of block



Scheme 1. Cross-Linking of Polyacetal Molecules with Epybromohydrine

size and state changes during the incubation, at (a) ambient temperature for 6 months, and (b) at 100 °C for 24 h. Media composition: 10-3 M HCl, pH ) 1; 50 mM sodium phosphate buffer, pH ) 2; 50 mM acetate buffer, pH ) 5; 50 mM tris-tricine buffer, pH ) 7.5; 50 mM borate buffer, pH ) 8.5. Derivatization of Carbonylpolyal PCF (II) via Reductive Amination with Ethylenediamine. (Aminoethyl)aminoPHF was prepared as a model intermediate for further amino group-targeted modifications. PCF solution (peak MW ) 200 kDa, broad MW distribution, 1 mg/mL in water, 100 mL) was incubated at 25 °C with ethylenediamine (1:10 mol/ mol to aldehyde groups) in the presence of cyanoborohydride (50% excess by amine) for 5 h at pH ) 8, and subsequently with a 50% excess of sodium borohydride for 2 h. The reaction mixture was neutralized with HCL and concentrated in an Amicon cell (10 kDa cutoff membrane), desalted on Sephadex G25, precipitated with ethanol, and dried. The amino group content was determined via trinitrobenzenesulfonate (TNBS) reaction.10 The product was fractionated by SEC to obtain fractions with MW from 15 to 300 kDa, Mw/Mr ) 2-2.5. [111In]DTPA-Labeled Amino-PHF. This procedure was developed, as an alternative to direct PHF acylation with DTPA, to achieve a higher degree of polymer modification with DTPA and higher label stability in vivo. Fractionated (aminoethyl)amino-PHF was prepared as described above. Acylation was carried out in 0.75 M Na2.7DTPA (pH ) 7.5), at a polymer concentration of 10 mg/mL, in the presence of a 10-fold excess of N-ethyl-N′-(N-diethylaminopropyl)carbodiimide (EDC). The reaction mixture was incubated overnight at ambient temperature, followed by purification on Sephadex G-25, labeling with 111In as described above, and subfractionation by HPSEC. Random-Point PHF Graft Copolymers with Poly-Llysine (Backbone). Graft copolymers were prepared to study the effect of the protective PHF side chains on polylysine backbone biokinetics. Polylysine hydrobromide (50 kDa) was modified with DTPA (15% of lysine residues) as described earlier,11 in 0.75 M Na2.7DTPA (pH ) 7.5) in the presence of EDC at 0 °C. Without separation, DTPA-polylysine backbone was conjugated with multiple PCF chains following a previously developed procedure.11 A 400-fold molar excess of PCF (MW fraction 5-10 kDa) in the form of a 15% solution was mixed with the above reaction mixture containing the modified poly-L-lysine. Sodium cyanoborohydride, 2-fold excess versus polylysine aminogroups, was added to the mixture, and conjugation was carried out overnight at ambient temperature. The product was reduced

with an excess of sodium borohydride. The resultant graft copolymer was isolated by ultrafiltration using an Amicon cell with a YM-100 membrane (cutoff: 100 kDa) with HPSEC control and lyophilized. Graft copolymers of polylysine and dextran B-512 with various oxidation/ring cleavage degrees were prepared analogously. Initially, a graft copolymer of DTPA-polylysine (50 kDa) and dextran (10 kDa) was synthesized, as described above, via conjugation of polylysine with a 400:1 molar excess of 10% periodate oxidized dextran in the presence of cyanoborohydride. The resultant graft copolymer was isolated by flow dialysis and lyophilized. The above dextran graft copolymer was then dissolved in water (100 mg/mL) and distributed, in equal 100 µL aliquots, to test tubes containing various amounts of 10% sodium periodate (0%, 20%, 50%, 100%, 200%, and 300% to glucose monomer). After 10 h of incubation at ambient temperature, all graft copolymers were reduced with sodium borohydride and isolated by gel chromatography on Sephadex G25. Carbohydrate content was determined using the phenol-sulfate method.12 For animal experiments, all graft copolymers were labeled with 111In and purified by HPSEC, as described above. In Vivo Studies. PHF and derivatives were studied in vivo to characterize polymer toxicity, in vivo kinetics, and biodistribution. Studies were performed in accordance with institutional guidelines. Unless otherwise stated, n ) 6 per group. Sprague-Dawley CD rats were used in biokinetics/ biodistribution and imaging studies. Male CD-1 mice were used in toxicity studies. Throughout the experimental procedures, rats and mice were anesthetized with 35 mg/kg sodium pentobarbital (intraperitoneally). Halothane (inhalation) was given as a supporting anesthetic when necessary. Rabbits were anesthetized with 10 mL of Ketamin/1.5 mL of Rompun. Intravenous injections were made through the tail vein (mice and rats) or marginal ear vein (rabbits). The initial phase of biokinetics was investigated by dynamic gamma-scintigraphy. Anesthetized animals were placed on a gamma camera collimator positioned horizontally. Dynamic image acquisition protocol was activated immediately prior the injection. Blood samples were collected via veins contralateral to the injection. Animals with significant extravasation at the injection point (as determined either visually or by γ-scintigraphy) were excluded from the experiment. Upon completion of the studies, the animals were euthanized with 100 mg/kg pentobarbital IV. PHF Toxicity. Underivatized PHF of high molecular weight (160 kDa) was used in the toxicity study to ensure minimal renal excretion of the polymer (the latter would

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Scheme 2. Physical Arrangement of Graft Copolymer Moleculesa

a

Graft copolymer interactions in vivo are defined by the structures present in the side chains.

mask possible toxic effects). Dose elevation in a limited group of animals showed no animal deaths at PHF doses up to 2 g intravenously and intraperitoneally;13 therefore, the larger 2 and 4 g/kg doses were studied in larger groups. Polymer solutions in 0.9% NaCl were sterilized by filtration through 0.22 µm polycarbonate membrane and injected intravenously. The larger dose (4 g/kg) was administered in two equal 2 g/kg injections with a 48 h interval to avoid administering an excessively high volume during a short period of time. Animal status and behavior were observed with weight registration daily for 30 days 1 h after the injection, then weekly for 120 days. Biokinetics of 111In-Labeled PHF. This experiment was designed to determine the rate of PHF clearance from blood and accumulation in the reticuloendothelial system (RES). 111In-labeled 30 and 500 kDa DTPA-aminoethylamino-PHF fractions were injected intravenously in rats at 1 mg/kg. The initial (16 min) in vivo kinetics was monitored by dynamic γ-scintigraphy. Static scintigraphy was then performed hourly to determine the optimal time for biodistribution studies. Blood samples were collected and radioactivity measured to determine blood clearance rates. Animals were euthanized at 20 h (rats injected with the low molecular weight polymer) and at 75 h (rats injected with the high molecular weight preparation). Tissue samples were collected, weighed, and studied on a gamma counter. Graft Influence on Graft Copolymer Biokinetics. The original hypothesis of this study suggested that lateral cleavage (or opening) of carbohydrate rings in a polysaccharide should result in reduced in vivo reactivity, which is expressed as longer circulation time and lower polymer accumulation in RES. To investigate whether ring cleavage would result in a longer circulation time and lower uptake in RES, we prepared a set of model preparations of essentially the same structure and size but with varying intact ring content. These preparations were assembled as soluble graft copolymers (physically, 15-20 nm nanoparticles) based on DTPA-modified polyL-lysine backbone. The backbone was surrounded by a dense brush formed by the side chains (Scheme 2). In the above structure, the side chains are shown in a somewhat stretched conformation; in fact, they are coiled and the backbone interactions with cells are thus sterically hindered. Graft copolymer interactions in vivo are thus defined by the structure of the side chains, interactions of which with cells are amplified, as compared to a single chain, by cooperative binding effects.14,15 In the synthesized graft

copolymers, the side chains were attached to the backbone via aliphatic amine bonds, and the label (111In-DTPA) via amide bond; both are stable in vivo. Radiolabeled graft copolymers, prepared, labeled, and purified as described above, were injected intravenously at 1 mg/kg. Blood clearance rates and biodistributions were studied as described above. Results Synthesis. The main objective of our synthetic studies was to develop a technique for lateral cleavage of carbohydrate rings in polysaccharides with complete cycle opening but without significant fragmentation of the main chain. The synthetic technique for producing acyclic semisynthetic polyals (see Materials and Methods) was developed on the basis of the following observations. (I) Excessively high temperature at either stage was found to decrease the yield of high molecular weight products. (II) At the oxidation stage, the pH was set by periodate (pH ) 2-3) with no need for adjustment. Higher pH in fact led to a higher fragmentation degree and incomplete ring cleavage. (III) The yield at the borohydride reduction stage was low if borohydride solution was added to the intermediate polymer, probably as a result of a pH shift to ca. pH ) 10. When the order of mixing was reversed, the yields significantly increased (most likely, for kinetic reasons). The optimized synthetic technique enabled consistently reproducible high yields of PHF with molecular weight distributions (as determined by SEC) only slightly shifted toward lower molecular weights, as compared to the precursor dextrans (e.g., see Figure 5). The shift could be related to either partial degradation of the main chain, or increased flexibility of the molecule (smaller segment length), or cleavage of side chains, or a combination of the above reasons. With many preparations of dextran B-512, the optimized procedure afforded 85-90% yields, while some dextran lots (most frequently “technical grade” preparations) gave lower, 40-50% yields with significant depolymerization. Oxidation of other polysaccharides resulted in lower yields and significantly depolymerized products, exceptions being levan from Erwinia Herbicola (2f6-poly-β-fructose) and inulin (2f1-poly-β-fructose). Inulin and levan gave relatively high yields of the respective polyketals (ca. 30% and 70%, respectively) (Scheme 3). In Scheme 3, the estimated polymerization degrees (n) are ca. 30 and 900 for the inulin- and levan-derived polyketals, respectively.


Figure 3. 1H NMR spectra (DMSO-d6:D2O 95:5) of PHF prepared from dextran B512 (Mn 188 000 Da). (A) Controlled incomplete oxidation, contains 19% vicinal glycol (uncleaved C3) at δ 4.49 ppm, d C1-H. (B) PHF prepared by exhaustive periodate oxidation, contains single triplet of C1-H at δ 4.62 ppm, J ) 5.2 Hz.

Figure 4. PHF hydrolysis: pH-dependence. The kinetics of median molecular weight reduction as determined by size exclusion HPLC. Dot sizes reflect standard deviations.

Precipitation of hydrophilic polyals from aqueous media was ineffective as a method of polymer isolation due to a high residual polymer content in the supernatant. Flow dialysis (ultrafiltration) was found to be a satisfactory purification technique for the intermediate carbonylpolyals and high molecular weight (MW > 150 kDa) hydroxypolyals. Retention of low molecular weight hydroxypolyals was unsatisfactory, resulting in high process losses (e.g., up to 90% for a 50 kDa polymer on a 30 kDa filter). Employing lower MW cutoff filters increased the retention but required significantly lower filtration rates, suggesting gel chromatography as an alternative purification technique. Chromatography on Sephadex G-25 and Sephacryl S-100 afforded nearly 100% product recovery (the latter with fractionation) for all studied molecular weights. However, due to the high viscosity of high MW polymers, their


chromatography can only be performed in highly diluted solutions, which complicates the subsequent polymer recovery. Therefore, for products with molecular weights of above ca. 100 kDa, flow dialysis remains the method of choice. The higher (practically quantitative) recovery of carbonylpolyals in flow dialysis procedures, as compared to fully reduced hydroxypolyals, can be explained by the observed significant reversible aggregation of these polymers in aqueous media (data not shown), up to gelling at high concentrations. Isolation of very low molecular weight carbonylpolyals (MW < 10 kDa) was performed on Sephadex G-25 following periodate inactivation with glycerol. All purified products were lyophilized. Because some acyclic polyals (notably PHF) were found to be hygroscopic, all lyophilized products were handled and stored under dry nitrogen. Versions of PHF containing pendant glycol groups were obtained by complete ring opening with incomplete (9597%) C3 elimination. Interestingly, the residual C3 seems to locate almost exclusively at C2 (Figure 3), as in V (Figure 2). The literature data suggest that in a cyclic carbohydrate the C2-C3 bond is cleaved 7-fold as fast as the C3-C4 bond, resulting mostly in IIb (Figure 2). The discrepancy is probably due to faster cleavage kinetics in IIb than in IIa. Properties. All intermediates (carbonylpolyals) and completely reduced polyals were obtained as colorless solids. Chemical analysis of lyophilized PHF (by Galbraith Laboratories) showed 44.19% carbon, 45.93% oxygen, 7.73% hydrogen, trace amounts of iodine (

Semisynthetic Hydrophilic Polyals - Biomacromolecules (ACS

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