Bioconjugate Chem. 1990, 1, 425-430
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Carrier Design: Biodistribution of Branched Polypeptides with a Poly(L4ysine) Backbone J. A. Clegg,' F. Hudecz,' G. Mezo,+M. V. Pimm, M. Szekerke,+and R. W. Baldwin Cancer Research Campaign Laboratories, University of Nottingham, University Park, Nottingham NG7 2RD, UK, and Research Group for Peptide Chemistry, Hungarian Academy of Science, L Eotvos University, Budapest 112, POB 32, Hungary 1518. Received September 5, 1990
The biodistribution has been examined in mice of a range of synthetic branched polypeptides which are based on a polylysine backbone but which differ in ionic charge, side-chain structure, and molecular size. Polycationic polypeptides, regardless of their size or primary structure at the branches, were cleared rapidly from the circulation, the liver being the major site of clearance. Polypeptides with glutamic acid in the side chain, which would be amphoteric under physiological conditions, showed a significantly prolonged blood survival, and this was seen with polypeptides in the range of molecular weights of 46 000 up to 213 000. Such polypeptides provide a useful system with which to investigate the effect of structural parameters on the pharmacokinetic properties of carrier molecules and would allow the selection of candidate carriers for a variety of uses.
INTRODUCTION
a
The use of macromolecular carriers for small molecules such as drugs has a wide range of applications, particularly in the field of drug delivery (1,2). Promising results have been reported concerning the alteration of the pharmacokinetics of biologically active compounds by their conjugation to macromolecules (3, 4 ) . In immunology, protein carriers are frequently applied to induce immune responses against covalently attached low molecular weight, nonimmunogenic epitopes, for monoclonal antibody production or for synthetic vaccine construction (5, 6). Conjugates of radionuclides and fluorophores to macromolecules could be useful in the development of biosensors and of various diagnostics. Based on results with synthetic branched polypeptides, (7, 8) a model system was established to gain information for the rational design of macromolecular carriers with desired characteristics required for specific purposes. A new group of branched polypeptides was synthesized in order to investigate systematically chemical (charge, size, primary structure, conformation) (9,10,1I) and biological (toxicity, immunogenicity, immunomodulatory potential, pyrogenicity, biodegradation) (12,13,14) parameters for an optimal carrier function (15). As an extension of this line of research and in respect of the possibility of in vivo application of these synthetic compounds (and their conjugates),the present studies were designed to elucidate correlations between the structural features of branched polypeptides and their biodistribution profile. We examined the blood clearance, wholebody survival, and tissue distribution of branched polypeptides corresponding to the general formula poly[Lys(xi-~~-Ala,)] or poly[Lys-(~~-Ala,-Xi)].These polypeptides are composed of a poly (L-LYS) backbone and of short side chains containing approximately three DL-Ala amino acid residues and one other ) amino acid residue. The schematic presentation of these structures is shown in Figure 1. In order to dissect the effect of size from that
(X
* To whom all correspondence should be addressed. +
L Eotvos University.
1043-1802/90/2901-0425$02.50/0
b oligo(DL-Ala) oligo(DL-Ala)
A
I
I
POlY(LY9)
PolYWYr)
Figure 1. Schematicpresentation of branched polypeptideswith poly(~-Ly~) backbone: (a)poly[Lys-(Xi-~~-Ah,)] (XAK),(b) poly-
(AXK). [Lys-(~~-Ala,-Xi)]
of other molecular characteristics, two groups of such polypeptides with basically identical side-chain composition, but with different size, were studied. In view of the importance of the side-chain structure in a-helix formation (9,II) or in immunological properties (12,13), we measured the biodistribution of polypeptides containing (i) amino acid residues of different identity (e.g., Leu, Pro, or Glu) at the side-chain terminal position, (ii) amino acid residues or D-G~u, L-Leu of different absolute configuration (L-G~u or D-Leu), (iii) amino acid residue at the side-chain end or at the position next to the polylysine backbone. These comparative studies were used to identify factors which could influence the biodistribution of the branched polypeptides.
X
EXPERIMENTAL PROCEDURES Abbreviations used in this paper follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature (16)in accord with the recommended nomenclature of graft polymers (I7). Branched polypeptides were synthesized as previously described (I1,18,19). Briefly, poly(Lys) was prepared by the polymerization of Na-carboxy-N~-(benzyloxycarbonyl)lysine anhydride under conditions that allowed an average degree of polymerization of either ca. 80-120 or ca. 4000 1990 American Chemical Society
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Clegg et al.
Bioconjugate Chem., Vol. 1, No. 6, 1990
500. After cleavage of the protecting groups, either poly[Lys-(~L-Ala,)](AK) was prepared by grafting of short oligomeric DL-Ala side chains onto the t-amino groups of poly(Lys) or benzyloxycarbonyl-protected leucine was coupled by the active-estermethod. Poly[Lys-(Xi-~~-Ala,)] (XAK) was synthesized by reacting a suitably protected amino acid pentachlorophenyl ester to the a-amino groups of AK. Blocking groups were removed completely with HBr in glacial acetic acid, as confirmed by UV spectroscopy at 254 nm. Poly[Lys-(~~-Ala,-Xi)] (AXK) was prepared by the introduction of DL- Ala oligomers to the previously deprotected a-amino and c-amino groups of poly[Lys-(Le~i)]or poly[Lys-(Glui)] by the aid of N-carboxy-DL-alanine anhydride. The primary structure of polypeptides were studied by amino acid analysis, by the identification of the branch-terminating amino acid residue (191, and by the determination of the enantiomer composition of the side chains (20). The size of these compounds was analyzed by sedimentation and gel chromatography (18,21). R a d i o i o d i n a t i o n of B r a n c h e d P o l y p e p t i d e s . Branched polypeptides were labeled (35-40 5% efficiency) with [1251]-N-succinimidy13-(4-hydroxyphenyl)propionate (Amersham International plc, Amersham, Bucks) using Bolton and Hunter’s procedure (22). Reagent solution (1020 pL) was added to plastic Eppendorf tubes and evaporated to dryness under a stream of nitrogen. Then 500 pL of polypeptide solution at 1 mg/mL in 0.1 M borate buffer (pH 8.6) was reacted with iodinated ester (2.5 mol of ester/mol of polypeptide). The reaction was allowed to proceed for 20 min at 0 OC and terminated by adding 500 pL of 0.2 M glycine in the same buffer for 5 min at 0 O C . The lz5I-labeledpolypeptide was purified on a G-25 Sephadex gel column using 0.066 M phosphate buffer (pH 7.6) containing 0.25% gelatin as eluent. Electrophoresis on native polyacrylamide gel with a continuous 8-2576 gradient (PhastGel gradient 8-25 Pharmacia-LKB, Uppsala, Sweden) was applied to assess the low molecular weight labeled product content of the preparation. Blood-Clearance and Tissue-Distribution Studies. Balb/c mice (female, 6-8 weeks old, Bantin and Kingman, Hull, UK) were used throughout these studies. Drinking water was supplemented with 0.1 % w/v sodium iodide. Groups of mice ( n = 3) received a single injection (0.2 mL) of 1251-labeledpolypeptide (8 MBq/mg, 50-200 pg/ kg) via tail vein. Serial blood samples (10 pL) were taken from the tail tip into microcapillary pipets (Drummond Microcaps, Drummond Scientific Co, Broomhall, PA), up to 6 h after injection. At this time the mice were killed and dissected. The blood samples, visceral organs, and residual carcasses were weighed and assayed for radioactivity in a conventional y-counter. Results of the bloodclearance study were expressed as a percentage of the zerotime count rate assuming the blood volume of the mouse (mL) to be 11.2% of the body weight (g) (23). Area under the blood concentration-time curve up to 6 h following injection was calculated by the trapezoidal rule ( 2 4 ) . Results of the tissue-distribution analysis were expressed as (i) percentage of the injected dose of radioactivity per gram of tissue or blood and (ii) ratios of radioactivity per gram of tissue to radioactivity per gram of blood (tissue to blood). Levels of statistical difference between groups of animals were assessed by Student’s t test. RESULTS AND DISCUSSION
Chemical Characteristics. A new type of branched polypeptide with poly(L-lysine)backbone was used in these studies (11,18,19). These polypeptides contain short side
chains composed of about three DL-alanine residues and one other amino acid residue (X) either at the end of the branches [poly[Lys-(Xi-o~-Ala,)],XAK] or at the position next to the polylysine backbone [p~ly[Ly~-(D~-Ala,-Xi)], AXK], where m 3 and i 4 1. These polypeptides represent a significantly modified version of multichain polypeptides used for immunological investigations by Sela et al. (25). In order to provide a simple, but versatile model system suitable for primary structure-conformation and chemical structure-carrierfunction analysis, the length of the poly(DL-Ala) side chains has been markedly shortened, and instead of copolymers, single amino acids were introduced into the branches. Due to the limited solubility of branched polypeptides containing even only short side chains of L-Ala or D-Ala, racemic oligo(DL-Ala) grafts were applied. Depending on the identity of the branch-terminating amino acid residue, these compounds have predominantly a-amino groups and express polycationic character (e.g., X = Leu, D-Leu, or Pro) or have a-amino groups as well as y-carboxylic groups and proved to be amphoteric under physiological conditions (e.g., Glu or D-G~u).The size of these branched polypeptides was defined by the average M,, Mw),the relative molar mass relative molar masses (Mn, distribution (Mz/Mw),and the average degree of polymerization (DP,) determined by applying sedimentation analysis and gel chromatography. It was found that all polymers investigated possess a fairly narrow distribution of relative molar mass (21). We have attempted to dissect the effect of size from that of other molecular characteristics and therefore two groups of such polypeptides with almost identical side-chain composition were prepared. The primary structure of these polypeptides was characterized by their amino acid composition and by the identification of the branch-terminating amino acid residue using HPLC analysis of the hydrolysates of dansylated polypeptides (20). The enantiomer composition of the side chains determined by reverse-phase HPLC and precolumn derivatization with Marfey’s reagent indicated that no stereospecific or stereoselective polymerization took place during the synthesis and the coupling of D- or L-amino acid to the poly[Lys-(~~-Ala,)] (AK) backbone did not result in racemization (26). Conformational properties of polypeptides, studied by circular dichroism (CD) spectroscopy, showed significant differences correlating with the identity, hydrophylic or hydrophobic nature, and configuration of the side-chainterminating amino acids and to the sequential order of amino acid residues in the side chains (9, 11, 27, 28). Chemical characteristics of the synthetic polypeptides are summarized in Table I. At pH 7.3 in 0.2 M NaCl the CD spectra of polypeptides with oligo(DL-Ala)branches correspond to unordered spatial arrangement. Very similar CD properties have been observed in case of Pro-containing analogues (poly[Lys(Proi-~~-Ala,)] (28). Polymers with L-Leu and D-Leu in the side chain terminal position, which also have polycationic character, adopt helical or partially helical conformation, respectively (27). In the case of Leu polypeptides, reversal of the amino acid sequence in the side chains resulted in a pronounced change in the CD spectra at acidic pH. In spite of the fact that alk showed a stronger tendency to form ordered structure, under physiological conditions both polypeptides assume helical confirmation (11). Polycationic polypeptides used in this study could assume helical conformation (Leu-containing polymers) and random coil structure (AK/ak and Pro-containing polypeptides), while amphoteric polypeptides (L-G~u or
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Bioconjugate Chem., Vol. 1, No. 6, 1990 427
Biodistribution of Branched Polypeptides
Table I. Characteristics of Branched Polypeptides DolvDeDtide poly[Lys-(~~-Ala,)] poly[Lys-(~~-Ala,)] poly[Lys-(Leu,-~~-Ala,)] poly[Lys-(Leui-~~-Ala,)] poly[Lys-(~~-Ala,-Leui)] poly[Lys-(~-Leui-~~-Ala,)] poly[Lys-(Proi-~~-Ala,)] poly[Lys-(Proi-~~-Ala,)] poly[Lys-(Glui-~~-Ala,)] poly[Lys-(~-Glui-~~-Ala,)] poly[Lys-(Glui-~~-Ala,)] poly[Lys-(~~-Ala,-Glui)]
abbreviation0 AK ak LAK lak alk D-LAK PAK Pak EAK D-EAK eak aek
Lvs 1 1
1 1 1 1 1 1 1
1 1 1
molar ratio of amino acids DL- Ala, 3.1 2.94 3.1 2.94 2.9 3.1 3.1 2.94 3.1 3.1 2.94 2.67
Xib
DPn'
0.98 0.81 0.79 0.98 0.96 0.94 0.96 0.95 0.93 1.0
450 100 450 100 92 450 450 100 450 450 100 100
MWd
&5%) 156 700 34 OOO 206 600 42 800 38 900 206 600 198 700 42 800 212 600 212 000 45 800 44 610
a Based on one-letter symbols of amino acids. Capital and small letters denote the size of the polypeptides. X = Leu, D-Leu, Pro, Glu, or and of the sideD-G~u.Number average degree of polymerization. Calculated from the average degree of polymerization of PO~Y(L-LYS) chain composition.
loo
1b !z=TE? ~
--C-
EAK
-t-
Table 11. Biodistribution Parameters of Branched Polypeptides
polypeptide AK ak LAK lak alk D-LAK PAK Pak EAK D-EAK eak aek
area under curve (0-6 h), % calcd t = 0 cpmh, (mean f SD) 28.9 f 2.3 26.8 f 3.2 26.9 f 1.8 21.2 f 1.2 41.9 f 2.7
