122
Biochimica et Biophysica Acta, 1(~34(1990}122-124
Elsevier BBAGEN 20258
BBA Report
Acid-sensitive dissociation between poly(lysine) and histamine-modified poly(glutamate) as a model for drug-releasing from carriers in endosomes Wei-Chiang Shen Drug Targeting Research, Division of Pharmaceutics, School of Pharmacy, University of Southern California, Los Angeles, CA (U.S.A.)
(Received 15 December1989)
Key words: pH sensitivecomplex; Poly(aminoacid); Imidazole-carboxyl;Endosome; Transferrin
Histamine was coupled to poly(L-glutamate) (PLG) to give a copolymer, poly(glutamylhistamineglutamate) (PHG), with approx. 40% of carboxyl groups in PLG being modified. Unlike either poly(L-histidine) (PLH) or PLG, PHG precipitated only in buffers with pH between 4 and 5. A complex was formed between PHG and poly(L-iysine) (PLL) at pH 7, but it was rapidly dissociated at pH 5 or lower. When PHG-linked transferrin (Tf-PHG) was used to deliver a PLL-conjugated [3H]methotrexate ([3H]MTX-PLL) in K562 leukemia cell cultures, an intracellnlar accumulation of the radioactivity was observed. These results suggest that a copolymer with both imidazole and carboxyl groups can be useful in the design of acid-sensitive, carrier-mediated drug delivery systems.
Controlled release of biologically active molecules from carriers in response to the change of environmental pH has drawn increasing attention to the areas of drug delivery and biotechnology. Acid-sensitive cleavage of covalent linkages in drug [1] or protein [2,3] conjugates has been applied successfully in many studies. An ideal acid-sensitive linkage should be ready to hydrolyze at very mildly acidic conditions, e.g., p H 5-6, and should be extremely stable at p H 7. None of the currently available linkages, however, can fulfill this requirement. For example, the cis-aconityl linkage is very stable at p H 7 with a half-life of more than 96 h, but the half-life at p H 5 is also as long as 6 h [1]. On the other hand, the ortho-ester linkage has a half-life of 10 min at p H 5, but its half-life at p H 7 is only 2 h [3]. The lack of a great sensitivity is understandable because a simple acid-catalyzed hydrolysis is a hydrogen ion-dependent reaction, and the hydrogen-ion concentrations differ only 10-fold in 1 p H unit. In many endocytotic pathways, however, the release of ligands from their receptors in the mildly acidic
Abbreviations: PLG, poly(L-glutamate); PHG, poly(glutamylhistamineglutamate; PLH, poly(L-histidine); PLL, poly(L-lysine); Tf, transferrin; MTX, methotrexate; EDC, 3-(3-dirnethylaminopropyl); PBS, phosphate-bufferedsaline.
Correspondence:W.C. Shen, Schoolof Pharmacy, PSC 404B, University of Southern California, Los Angeles, CA 90033, U.S.A.
endosomal environment, i.e., approx, p H 5.0 [4], is achieved through the non-covalent interaction. For example, asialoglycoproteins which bind tightly to the cell membrane receptors are readily dissociated in endosomes due to a conformational change of the receptor induced by the low pH after the internalization [5]. The dissociation takes place in a very short time, and essentially all uncoupled receptors return to the cell surface within 10 min [6]. Such a dissociation between two macromolecules seems to be extremely sensitive and efficient in response to a mild acidification. In this report, copolymers with both imidazole and carboxyl groups are proposed as pH sensitive carriers when complexed with polycationic polymers to mimic the ligand-receptor dissociation in endosomes. Since simple copolymers with this composition, e.g., poly(Glu-His) or poly(Asp-His), are not available at the present time, we chose the histamine-modified PLG, i.e., PHG, as a model to demonstrate the pH sensitivity and the potential applications. P H G was prepared by the following procedure: To a solution of 30 mg P L G ( M r = 43000, sodium salt; Sigma) and 25 mg histamine dihydrochloride in 2 ml of 0.05 M phosphate buffer (pH 5.5), 40 mg of 3-(3-dimethylaminopropyl)carbodiimide (EDC) was added in four portions with 5 min intervals. After the final addition of EDC, the reaction solution was stirred at 25 ° C for 1 h and then adjusted to p H 7 with 1 M NaOH. The final reaction solution was then dialyzed exhaustively at 4 ° C in phosphate-buffered
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
123 1.0
0 0 ~r
~ 0.5.u
S 12 3 4 5 6 7 8 pH
Fig. 1. pH-dependent solubility of different poly(amino acids). PLH (A), PLG (o) and PHG (e), were dissolved at 1 mg/ml separatelyin citrate-phosphate buffers with pH ranged from 2 to 8. Turdibity in each of the solution was measured as the absorbance at 400 nm.
saline, pH 7 (PBS). The imidazole content in the final conjugate was measured using a diazotization method [7]. In the P H G obtained 40% Glu had been modified, indicating the ratio of carboxyl to imidazole groups was 1.5:1. The solubilities of PHG, as well as P L G and poly(Lhistidine) (PLH, M r = 10000; Sigma), determined as the turbidity in the solution, were measured as the absorbance at 400 nm in citrate-phosphate buffers. As shown in Fig. 1, PLH and P L G were not soluble in buffers with p H values above 6 and below 3, respectively. However, the P H G solution showed turbidity only between p H 4-5. These solubility profiles are consistent with the p K a values of imidazole and carboxyl in PLH and PLG, i.e., 6 and 4, respectively. They also indicate the occurrence of two steps of charge changes in PHG, i.e., from p H 6 to 5 (polyanionic to neutral), and from pH 4 to 3 (neutral to polycationic). The fact that heavy precipitation was observed at p H 4 - 5 suggests that intramolecular charge complexes were formed in P H G under the mildly acidic conditions. The gross charge changes in P H G can provide two distinct p H sensitivities, i.e., from neutral to mildly acidic environment which will dissociated it from another polycationic polymer, and from strongly to mildly acidic environment, which will dissociate it from another polyanionic polymer. The change from a neutral (pH 7) to a mildly acidic environment (pH 5-6) is more relevant to the conditions in many drug delivery systems. For example, it has been found in the process of endocytosis where macromolecules internalized by mammalian cells are rapidly transported into mildly acidic compartments, i.e., endosomes [8]. Therefore, the complex formation between P H G and a polycationic polymer, PLL ( M r -- 52 000; hydrobromide) was studied in order to explore the applications of P H G in the promotion of the endosomal dissociation. The complex "ormation between P H G and PLL was determined by xing equal concentration (1 m g / m l ) of each polymer ,ffers with various p H values and the turbidity was
measured spectrophotometrically. Fig. 2 shows that at p H 6 or higher, P H G and PLL formed a stable complex as indicated by the heavy turbidity. Dissociation of the complex was observed when the solution was lowered to p H 6 and the complex was completely dissociated between p H 4 and 5 (Fig. 2). Dissociation between P H G and a of M r 3000 PLL at p H 4 could also be detected by a Sephadex G-50 gel filtration chromatography (data not shown). When P L G was used instead of PHG, no dissociation of the polyanion, polycation complex was observed until the p H was 3 or lower (Fig. 2). The potential application of P H G - P L L complexes in the pH-sensitive delivery of macromolecules to endosomes can be demonstrated in transferrin receptormediated uptake of an anticancer drug, MTX. It is known that transferrin is internalized by mammalian cells through receptor-mediated endocytosis [8]. After the ferric ions dissociate from transferrin in endosomes, apo-transferrin molecules are rapidly recycled with the receptors to the cell surface [8]. Due to the lack of intracellular degradation, drugs that directly conjugated to transferrin, as shown in 125I-labelled transferrin [9], are not expected to accumulate inside the cell. Since the dissociation of ferric ions from transferrin occurs at about p H 5.5 [10], the P H G . PLL complex may also dissociate in the same intracellular compartment. To demonstrate the dissociation of the complex system, we have prepared transferrin and P H G conjugates by coupling these two macromolecules with a thioether linkage. H u m a n transferrin (30 mg, diferric forms) was reacted with 1 mg of sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB, Pierce Chemical Co.) in 1.5 ml PBS. After 1 h at 25 ° C, the reaction mixture was dialyzed exhaustively at 4 ° C in PBS. Sulfhydrylcontaining P H G or P L G was prepared by first coupling 2 mg of cystamine (dichloride) to 30 mg of P H G or P L G by EDC, reduced with 20 m M of dithiothreitol, and subsequently purified by Sephadex G-50 gel filtration. The freshly prepared sulfhydryl-containing P H G
Biochimica et Biophysica Acta, 1(~34(1990}122-124
Elsevier BBAGEN 20258
BBA Report
Acid-sensitive dissociation between poly(lysine) and histamine-modified poly(glutamate) as a model for drug-releasing from carriers in endosomes Wei-Chiang Shen Drug Targeting Research, Division of Pharmaceutics, School of Pharmacy, University of Southern California, Los Angeles, CA (U.S.A.)
(Received 15 December1989)
Key words: pH sensitivecomplex; Poly(aminoacid); Imidazole-carboxyl;Endosome; Transferrin
Histamine was coupled to poly(L-glutamate) (PLG) to give a copolymer, poly(glutamylhistamineglutamate) (PHG), with approx. 40% of carboxyl groups in PLG being modified. Unlike either poly(L-histidine) (PLH) or PLG, PHG precipitated only in buffers with pH between 4 and 5. A complex was formed between PHG and poly(L-iysine) (PLL) at pH 7, but it was rapidly dissociated at pH 5 or lower. When PHG-linked transferrin (Tf-PHG) was used to deliver a PLL-conjugated [3H]methotrexate ([3H]MTX-PLL) in K562 leukemia cell cultures, an intracellnlar accumulation of the radioactivity was observed. These results suggest that a copolymer with both imidazole and carboxyl groups can be useful in the design of acid-sensitive, carrier-mediated drug delivery systems.
Controlled release of biologically active molecules from carriers in response to the change of environmental pH has drawn increasing attention to the areas of drug delivery and biotechnology. Acid-sensitive cleavage of covalent linkages in drug [1] or protein [2,3] conjugates has been applied successfully in many studies. An ideal acid-sensitive linkage should be ready to hydrolyze at very mildly acidic conditions, e.g., p H 5-6, and should be extremely stable at p H 7. None of the currently available linkages, however, can fulfill this requirement. For example, the cis-aconityl linkage is very stable at p H 7 with a half-life of more than 96 h, but the half-life at p H 5 is also as long as 6 h [1]. On the other hand, the ortho-ester linkage has a half-life of 10 min at p H 5, but its half-life at p H 7 is only 2 h [3]. The lack of a great sensitivity is understandable because a simple acid-catalyzed hydrolysis is a hydrogen ion-dependent reaction, and the hydrogen-ion concentrations differ only 10-fold in 1 p H unit. In many endocytotic pathways, however, the release of ligands from their receptors in the mildly acidic
Abbreviations: PLG, poly(L-glutamate); PHG, poly(glutamylhistamineglutamate; PLH, poly(L-histidine); PLL, poly(L-lysine); Tf, transferrin; MTX, methotrexate; EDC, 3-(3-dirnethylaminopropyl); PBS, phosphate-bufferedsaline.
Correspondence:W.C. Shen, Schoolof Pharmacy, PSC 404B, University of Southern California, Los Angeles, CA 90033, U.S.A.
endosomal environment, i.e., approx, p H 5.0 [4], is achieved through the non-covalent interaction. For example, asialoglycoproteins which bind tightly to the cell membrane receptors are readily dissociated in endosomes due to a conformational change of the receptor induced by the low pH after the internalization [5]. The dissociation takes place in a very short time, and essentially all uncoupled receptors return to the cell surface within 10 min [6]. Such a dissociation between two macromolecules seems to be extremely sensitive and efficient in response to a mild acidification. In this report, copolymers with both imidazole and carboxyl groups are proposed as pH sensitive carriers when complexed with polycationic polymers to mimic the ligand-receptor dissociation in endosomes. Since simple copolymers with this composition, e.g., poly(Glu-His) or poly(Asp-His), are not available at the present time, we chose the histamine-modified PLG, i.e., PHG, as a model to demonstrate the pH sensitivity and the potential applications. P H G was prepared by the following procedure: To a solution of 30 mg P L G ( M r = 43000, sodium salt; Sigma) and 25 mg histamine dihydrochloride in 2 ml of 0.05 M phosphate buffer (pH 5.5), 40 mg of 3-(3-dimethylaminopropyl)carbodiimide (EDC) was added in four portions with 5 min intervals. After the final addition of EDC, the reaction solution was stirred at 25 ° C for 1 h and then adjusted to p H 7 with 1 M NaOH. The final reaction solution was then dialyzed exhaustively at 4 ° C in phosphate-buffered
0304-4165/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
123 1.0
0 0 ~r
~ 0.5.u
S 12 3 4 5 6 7 8 pH
Fig. 1. pH-dependent solubility of different poly(amino acids). PLH (A), PLG (o) and PHG (e), were dissolved at 1 mg/ml separatelyin citrate-phosphate buffers with pH ranged from 2 to 8. Turdibity in each of the solution was measured as the absorbance at 400 nm.
saline, pH 7 (PBS). The imidazole content in the final conjugate was measured using a diazotization method [7]. In the P H G obtained 40% Glu had been modified, indicating the ratio of carboxyl to imidazole groups was 1.5:1. The solubilities of PHG, as well as P L G and poly(Lhistidine) (PLH, M r = 10000; Sigma), determined as the turbidity in the solution, were measured as the absorbance at 400 nm in citrate-phosphate buffers. As shown in Fig. 1, PLH and P L G were not soluble in buffers with p H values above 6 and below 3, respectively. However, the P H G solution showed turbidity only between p H 4-5. These solubility profiles are consistent with the p K a values of imidazole and carboxyl in PLH and PLG, i.e., 6 and 4, respectively. They also indicate the occurrence of two steps of charge changes in PHG, i.e., from p H 6 to 5 (polyanionic to neutral), and from pH 4 to 3 (neutral to polycationic). The fact that heavy precipitation was observed at p H 4 - 5 suggests that intramolecular charge complexes were formed in P H G under the mildly acidic conditions. The gross charge changes in P H G can provide two distinct p H sensitivities, i.e., from neutral to mildly acidic environment which will dissociated it from another polycationic polymer, and from strongly to mildly acidic environment, which will dissociate it from another polyanionic polymer. The change from a neutral (pH 7) to a mildly acidic environment (pH 5-6) is more relevant to the conditions in many drug delivery systems. For example, it has been found in the process of endocytosis where macromolecules internalized by mammalian cells are rapidly transported into mildly acidic compartments, i.e., endosomes [8]. Therefore, the complex formation between P H G and a polycationic polymer, PLL ( M r -- 52 000; hydrobromide) was studied in order to explore the applications of P H G in the promotion of the endosomal dissociation. The complex "ormation between P H G and PLL was determined by xing equal concentration (1 m g / m l ) of each polymer ,ffers with various p H values and the turbidity was
measured spectrophotometrically. Fig. 2 shows that at p H 6 or higher, P H G and PLL formed a stable complex as indicated by the heavy turbidity. Dissociation of the complex was observed when the solution was lowered to p H 6 and the complex was completely dissociated between p H 4 and 5 (Fig. 2). Dissociation between P H G and a of M r 3000 PLL at p H 4 could also be detected by a Sephadex G-50 gel filtration chromatography (data not shown). When P L G was used instead of PHG, no dissociation of the polyanion, polycation complex was observed until the p H was 3 or lower (Fig. 2). The potential application of P H G - P L L complexes in the pH-sensitive delivery of macromolecules to endosomes can be demonstrated in transferrin receptormediated uptake of an anticancer drug, MTX. It is known that transferrin is internalized by mammalian cells through receptor-mediated endocytosis [8]. After the ferric ions dissociate from transferrin in endosomes, apo-transferrin molecules are rapidly recycled with the receptors to the cell surface [8]. Due to the lack of intracellular degradation, drugs that directly conjugated to transferrin, as shown in 125I-labelled transferrin [9], are not expected to accumulate inside the cell. Since the dissociation of ferric ions from transferrin occurs at about p H 5.5 [10], the P H G . PLL complex may also dissociate in the same intracellular compartment. To demonstrate the dissociation of the complex system, we have prepared transferrin and P H G conjugates by coupling these two macromolecules with a thioether linkage. H u m a n transferrin (30 mg, diferric forms) was reacted with 1 mg of sulfosuccinimidyl 4-(p-maleimidophenyl)butyrate (sulfo-SMPB, Pierce Chemical Co.) in 1.5 ml PBS. After 1 h at 25 ° C, the reaction mixture was dialyzed exhaustively at 4 ° C in PBS. Sulfhydrylcontaining P H G or P L G was prepared by first coupling 2 mg of cystamine (dichloride) to 30 mg of P H G or P L G by EDC, reduced with 20 m M of dithiothreitol, and subsequently purified by Sephadex G-50 gel filtration. The freshly prepared sulfhydryl-containing P H G
