Preparation of micelle-forming polymer-drug conjugates - American

Feb 13, 1992 - micelles to sustained release of drugs (16, IT).Although. Ringsdorfs group suggested micelle formation of their polymer-drug conjugate ...
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Bioconjugate Chem. 1992, 3, 295-301

295

Preparation of Micelle-Forming Polymer-Drug Conjugates Masayuki Yokoyama,* Glenn S. Kwon, Teruo Okano, Yasuhisa Sakurai, Takashi Seto,f and Kazunori Kataoka*-* 1

Institute of Biomedical Engineering, Tokyo Women’s Medical College, Kawada-cho, 8-1, Shinjuku-ku, Tokyo 162, Japan, Nippon Kayaku Company, Ltd., Iwahana 269, Takasaki-shi, Gunma 370-12, Japan, and Department of Materials Science and Technology, Faculty of Industrial Science and Technology, Science University of Tokyo, Yamazaki 2641, Noda-shi, Chiba 278, Japan. Received February 13, 1992 Adriamycin, a hydrophobic anticancer drug, was conjugated with polyethylene oxide)-poly(aspartic acid) block copolymers composed of various lengths of each block copolymer segment ranging from 1000 to 12 000 in molecular weight and from 10 to 80 units, respectively. Conjugation was achieved without precipitation by adjusting the ratio of adriamycin to aspartic acid residues of the block copolymer and the quantity of DMF used for the reaction. Thus obtained conjugates showed high water solubility irrespective of a large amount of the conjugated adriamycin. Furthermore, these conjugates were found to form micellar structures with a hydrophobic inner core and a hydrophilic outer shell. This micellar architecture may be utilized for effective drug targeting.

-w

INTRODUCTION

Drug targeting using drug carriers is a very attractive “target” both for basic sciences and medical applications. The ideal drug is expected to act only at the designated target site of the drug and not at the other sites and, consequently, to express specificity and high activity without toxic side effects. To realize this ideal drugtargeting system, several types of drug carriers (e.g., microspheres, liposomes, and polymeric carriers) have been investigated especially for anticancer drugs. However,

:

micelle

formation

O: drug

hydrophilic segment

^ hydrophobic segment

successful practical applications have not yet been obtained mainly due to the nonselective scavenging by normal cells even in the case of monoclonal antibodies (1-4). It is expected that drug targeting by carriers cannot succeed

biodistribution, pharmacokinetics

without maintenance of their stable circulation in blood.

For microspheres (5) and liposomes (6), nonspecific scavenging by the reticuloendothelial system is the main obstacle to effective drug targeting unless these microspheres and liposomes were appropriately modified (5, 7, 8). For polymeric carriers, low water solubility of drugpolymer conjugates often causes problems in their synthesis (9-11) and in their injection into the blood stream (12). Since most drugs have a hydrophobic character, conjugation of the drugs with a polymer easily leads to precipitation because of the high, localized concentration of hydrophobic drug molecules bound along the polymer chain. The authors have been investigating polymeric micelles as an alternative drug-carrier system (13-15). An ABtype block copolymer composed of hydrophilic and hydrophobic components can form a micellar structure as a result of its amphiphilic character as illustrated in Figure 1. The hydrophobic drug-binding segment forms the hydrophobic core of the micelle, while the hydrophilic segment surrounds this core as a hydrated outer shell. With a core-shell structure, polymeric micelles may maintain their water solubility by inhibiting intermicellar aggregation of the hydrophobic cores irrespective of high hydrophobicity of the inner cores. That is, polymeric micelles can utilize the hydrophobicity of the drug-binding segment, which causes precipitation in conventional *

f 1

Author to whom correspondence should be addressed. Nippon Kayaku Co., Ltd. Science University of Tokyo. l043-1802/92/2903-0295$03.00/0

pharmacological activity

Figure

1.

Concept of

a

micelle-forming polymeric drug.

a suitable structure for drug the functions which are required Furthermore, targeting. for drug carriers can be shared by the structurally separated segments of the block copolymer. The outer shell is responsible for interactions with the biocomponents such as proteins and cells. These interactions may determine pharmacokinetic behavior and biodistribution of drugs, therefore, in vivo delivery of drugs may be controlled by the outer shell independently of the inner core of the micelle which expresses pharmacological activities. This heterogeneous structure is more favorable to construct highly functionalized carrier systems than the conventional polymeric carrier systems. Until now, few studies were done to focus on the application of polymeric micelles to drug carriers. Ringsdorf et al. reported an idea of application of polymeric micelles to sustained release of drugs (16, IT). Although Ringsdorfs group suggested micelle formation of their polymer-drug conjugate on the basis of the data from dyesolubilization and retardation in release rate of the bound drug, they did not confirm micelle formation by more direct methods. Kavanov et al. (18) reported an increase in in vivo activity of a drug associated with a polymeric amphiphile. Kavanov, however, did not distinguish the effect of formation of a polymer-drug conjugate on efficient

polymeric drugs, to achieve

©

1992 American Chemical Society

296

Bioconjugate Chem., Vol. 3, No. 4, 1992 HN

CH 3 -(OCH 2 CH 2

^

NH 2

+

Yokoyama et al.

3-[3-(dimethylamino)propyl]carbodiimide (EDC) was pur-

//

c:

—

chased from Peptide Institute, Inc., Osaka, Japan,

>

I

0

I

CH2COOCH2@ 1,

CHj-PEO-NH: 2,

BLA-NCA

CH3-(OCH2CH2^NH-(COCHNH)^-H

CH2COOCH2^^ 3,

PEO-PBLA

debenzylation

-

CHs-(OCH2CH2tNHiCOCHNH>—(COCH2CHNH)— H I

CH2COOH

COOH

4, PEO-P(Asp)

ADR h CH3HOCH2CH2)rNH-(COCHNH)—(COCH2CHNH)— y I

I

CH2COR

O

Figure

2. Synthesis

COR

OH

of PEO-P(Asp(ADR)).

delivery to the target from that on an increase in permeability of biological membranes given by the polymeric amphiphile, and that study did not present evidence for micelle formation in a physiological condition. We have been studying polymeric micelles to realize a drugcarrier system stably existing under in vivo conditions for a long time period, and we directly confirmed micelle formation in vitro (13,14) and in vivo (19) with adriamycinconjugated polyethylene oxide)-poly(aspartic acid) block copolymer [PEO-P(Asp(ADR))1 by dynamic laser light scattering and gel-filtration chromatography. Furthermore, this micelle-forming polymeric drug was shown to express significantly higher in vivo anticancer activity than free adriamycin (ADR) against P388 murine leukemia (14) and several solid tumors such as murine adenocarcinoma C 26 (19). The chemical structure of the micelle-forming polymeric drug synthesized by the authors previously (13, 20) is shown in Figure 2. One segment of the block copolymer is polyethylene oxide), which becomes the outer shell of the micelle, and the other segment is poly(aspartic acid), which becomes the inner core after binding a hydrophobic anticancer drug, ADR. This paper describes preparations of various compositions of this micelle-forming polymeric anticancer drug [PEO-P(Asp(ADR))] by changing chain lengths of both the polymer segments. Evidence for micelle formation of these PEO-P(Asp(ADR))s is presented. ****]

EXPERIMENTAL PROCEDURES

Chemicals. Adriamycin hydrochloride (ADR’HCl) was

purchased from Sanraku Inc., Yatsushiro, Japan. 1-EthylAbbreviations used: EDC, l-ethyl-3-[3-(dimethylamino)propyllcarbodiimide; ADR, adriamycin; ADR-HC1, adriamycin hydrochloride; Asp, aspartic acid; BLA-NCA, 0-benzyliV-carboxyL-aspartate anhydride; PEO, polyethylene oxide); PBLA, poly(0-benzyl L-aspartate); P( Asp), poly (aspartic acid); PEO-PBLA, polyethylene oxide)-poly(0-benzyl L-aspartate) block copolymer; PEO-P(Asp), poly (ethylene oxide)-poly (aspartic acid) block copolymer; PEO-P(Asp(ADR)), adriamycin-conjugated polyethylene oxide)-poly(aspartic acid) block copolymer. 1

a-

Methyl-w-aminopoly(oxyethylene) (CH3-PEO-NH2, 1 in Figure 2) was purchased from Nippon Oil & Fats Co., Ltd., Kawasaki, Japan. 0-Benzyl JV-carboxy-L-aspartate anhydride (2) was purchased from Kokusan Chemical Works, Ltd., Tokyo, Japan. Other chemicals were of reagent grade and were used as purchased unless otherwise stated. General Procedures. XH-NMR spectra were obtained in CDCI3 (PEO-PBLA) or D2O (PEO-P(Asp)) with a Varian Gemini-500 NMR instrument. HPLC was carried out using a JASCO HyPer LC-800 system (Tokyo, Japan) at a flow rate of 1.0 mL/min at 40 °C with a Shodex OHpak KB-802.5 column and a KB-803 column in 0.1 M phosphate-buffered solution (pH 7.4) for PEO-P(Asp) and with an Asahipak GS-520 H column in 0.1 M phosphatebuffered solution (pH 7.4, containing 0.3 M NaCl) for PEOP(Asp(ADR)). One hundred microliter samples were injected into the columns at a concentration of 0.2 wt % in distilled water for PEO-P(Asp) and 20 ng ADR-HC1 equiv/mL in phosphate-buffered saline (Na/Na, pH 7.4, 0.155 M) for PEO-P(Asp(ADR)). The detection of the polymers was performed by refractive index (with a JASCO 830-RI detector) for PEO-P(Asp) or absorption at 485 nm (with a JASCO 870-UV detector) for PEO-P(Asp(ADR)). Laser scattering measurements on the conjugate solutions were carried out using a Photal dynamic laser scattering spectrometer DLS-700 (Otsuka Electronics Co. Ltd., Tokyo, Japan) with an argon laser beam at a concentration of 20 ng ADR-HC1 equiv/mL in phosphatebuffered saline (Na/Na, pH 7.4,0.155 M) at 25 °C, and the values are expressed in weight fraction and weight average. The synthetic route of PEO-P(Asp(ADR)) is shown in Figure 2. The procedure was based on the previously

reported one (14) with slight modifications. Abbreviations used for block copolymers were based on molecular weight of polyethylene oxide) (PEO) chain and polymerization degree of BLA (or Asp) units; for example, 1-10 means a block copolymer composed of a PEO chain of MW = 1000 and a PLBA or P(Asp) chain which has 10 units of BLA (or Asp).

Synthesis of Poly(ethylene oxide)-Poly(0-benzyl L-aspartate) Block Copolymer (PEO-PBLA). 0-Benzyl N-carboxy-L-aspartate anhydride (BLA-NCA, 2) was dissolved in doubly distilled N^V-dimethylformamide (DMF), followed by an addition of distilled chloroform. a-Methyl-w-aminopoly(oxyethylene) (CH3-PEG-NH2,1) was dissolved in distilled chloroform and added to the solution of 2. Quantities of the solvents in the reaction mixture were adjusted to 1.5 mL of DMF and 15 mL of chloroform per 1 g of BLA-NCA. The reaction mixture was stirred at 35 °C in a stream of dry nitrogen until BLANCA disappeared by the detection of characteristic peaks (1850 and 1790 cm-1) in an IR spectrum. Then, the reaction mixture was poured into a 10-fold volume of diethyl ether at 0 &C, and a precipitate was collected by filtration and washed with diethyl ether, followed by drying in vacuo. Synthesis of Polyethylene oxide)-Poly(aspartic acid) Block Copolymer (PEO-P(Asp)). Three-fold equivalents of sodium hydroxide to benzyl groups of the block copolymer was added to PEO-PBLA with 0.5 N NaOH aqueous solution. The reaction mixture was vigorously stirred at room temperature. When the reaction mixture clarified, the solution was neutralized with acetic acid, followed by dialysis against distilled water using a Spectrapor 6 didysis membrane (molecular weight cutoff = 1000) until low molecular weight contaminants such as acetic acid could not be detected by ^-NMR measure-

Preparation of Micelle-Forming Polymer-Drug Conjugates

Bioconjugate Chem., Vol. 3, No. 4, 1992

297

Table I. Synthesis of PEO-PBLA feed composition run

MW of PEO chain

MeO-PEO-NH2 (g)

BLA-NCA (g)

reaction time (h)

yield (g) [%]6

BLA units'

1-10“ 1-20 1-40 1-80 2-10 2-20 2-40 2-80 5-10 5-20 5-40 5-80 12-20 12-40 12-80

1000 1000 1000 1000 2000 2000 2000 2000 5000 5000 5000 5000 12000 12000 12000

2.01 1.51

5.01 7.53 4.98 4.98 3.12 6.23

14.0 17.5 26.0 27.5 13.5 14.5 38.0 38.5 26.0 21.5 24.0 26.0 17.0 20.5 22.5

5.31 [86.5] 6.52 [84.6]

10.4 22.6

4.18 [90.9] 3.95 [90.8] 4.33 [85.4] 7.01 [91.9] 4.65 [91.2] 4.15 [90.2] 5.13 [91.0] 11.73 [91.9] 4.82 [91.2] 4.74 [91.4] 4.82 [89.8] 6.08 [90.3] 5.18 [87.6]

40.0 73.3 9.6

0.50 0.25 2.50 2.50 1.00 0.50 4.00 7.01 2.00 1.21 4.00 4.00 2.50

4.98 4.98 1.99 7.00 3.99 4.83 1.66 3.32 4.15

'

19.4 38.3 72.0 9.0 19.3 38.6 75.3 19.5 38.0 73.5

0 Abbreviation was used based on molecular weight of the PEO chain and polymerization degree of BLA units; for example, 1-10 means block copolymer composed of the PEO chain of MW 1000 and the PBLA chain which has 10 units of BLA. 6 Percent yield was calculated with the weight of (CH3-PEO-NH2 + BLA-NCA x 0.823) as 100%.c Determined by !H NMR using methylene proton (OCH2CH2, 3.7 ppm) of the PEO chain and benzyl proton (CRgCgHg, 5.1 ppm) of the PBLA chain.

a

ment. Then, polyethylene oxide)-poly(aspartic acid) block copolymer [PEO-P(Asp), 4] was obtained by freezedrying. Binding of ADR to PEO-P(Asp). ADR-HC1 was mixed with DMF, followed by an addition of 1.3 equiv of triethylamine (with 10 v/v % solution in DMF) with vigorous stirring at 0 °C. PEO-P(Asp) (4), dissolved in distilled water, and l-ethyl-3- [3-(dimethylamino)propyl] carbodiimide (EDC) were successively added to the solution of ADR. The reaction mixture was stirred at 0 °C for 4 h, followed by the second addition of EDC. Then, the reaction mixture was stirred for 15 h at room temperature. The resulting solution was dialyzed overnight against 0.1 M sodium acetate buffered solution (pH 4.5, using approximately 100-fold volume dialysate) using a Spectrapor 6 membrane (MW cutoff = 1000). It seems that this transfer from an environment in an organic solvent to aqueous medium induces micelle formation of the drug-block copolymer conjugates with a hydrophobic drug-binding inner core and hydrophilic PEO outer shell. For several samples listed in Table IV, the resulting solution was further purified by ultrafiltration using a Minitan ultrafiltration system (Japan Millipore Ltd.) and Amicon ultrafiltration cells Model 8050 and 8200 (Grace). The content of ADR in the conjugate [PEO-P(Asp(ADR))> 5] was determined by measuring absorbance at 485 nm in DMF on the assumption that €485 of the ADR residue bound to the polymer was the same as that of free ADR. -

RESULTS

Synthesis of PEO-PBLA. Fifteen compositions of PEO-PBLA were synthesized with combinations of four lengths of the PEO chains and four lengths of the BLA units as summarized in Table I. Polymerizations of BLANCA in all the runs had progressed homogeneously in the mixed solvent of DMF and chloroform (1:10), and they were found to complete within 38.5 h by confirming the disappearance of the characteristic peaks of BLA-NCA in IR spectra. All samples were obtained in high yields over 85 %. The number of the BLA units in PEO-PBLA was determined by the peak ratio of methylene protons (OCH2CH2,3.7 ppm) of the PEO chain and benzyl protons (CH2C6H5, 5.1 ppm) of the PBLA chain in a ^-NMR spectrum measured in CDCI3. These numbers agreed well

with the theoretical values. Synthesis of PEO-P(Asp). The reaction mixtures became homogeneous within 2 h after an addition of the NaOH solution for all the runs except 2-80. Successful

debenzylation was confirmed by ^-NMR spectroscopy in D20. No residual benzyl groups were detected in the obtained polymer samples, and the numbers of the Asp units in the PEO-P(Asp) chain determined by the peak ratio of methylene protons (3.7 ppm) of the PEO chain and methylene protons (2.8 ppm) of the P(Asp) chain were identical to the numbers of BLA units of the corresponding PEO-PBLA within experimental error of the NMR measurement. PEO-P(Asp) were obtained in high yields over 90% for most runs. Yields were found to exceed 100% for some runs. That was likely due to incorporated sodium as sodium salts in carboxyl groups of the P(Asp) chain, and due to some water that could not be removed even after lyophilization. It is known that a-amide bonds of the BLA units were transformed into /3-amide bonds by alkaline hydrolysis to a considerable degree (21). The content of the /3-amide bond was found to be 75 mol % among all the amide bonds in the P(Asp) chain of PEOP(Asp) 5-20 by measuring a peak ratio of methine protons of a-amide (4.66 ppm) and /3-amide (4.47 ppm) of a 1HNMR spectrum in D2O at pD 9.2. For other samples, contents of the /3-amide bond were not measured; however, these contents seem to be around 75 mol % independent of their compositions, since it was reported (23) that 75 mol % of the /3-amide bond was also found in P(Asp) homopolymer obtained from PBLA homopolymer (degree of polymerization = 110) by an alkaline hydrolysis procedure. The molecular weight distribution of PEO-P(Asp) was evaluated by gel-filtration chromatography as summarized in Table II. Two peaks were observed for block copolymers 1-10 and 2-10, while the other block copolymers possessed one peak in their chromatograms, as typified by PEOP(Asp) 2-10, 2-20, and 2-40 in Figure 3. This heterogeneity is considered to result from an acceleration effect of NCA polymerization above about a degree of polymerization of eight (22). It was reported (23) that elongation of 7-benzyl L-glutamate units in poly(7-benzyl L-glutamate) chains composed of over about 8 units progressed more rapidly in dioxane than the polymer chains not reaching this polymerization degree. It is expected that such bimodal molecular weight distribution was obtained with a valley which corresponds to PEO-P( Asp) with about eight Asp units. For the block copolymers with 20, 40, and 80 Asp units, unimodal molecular weight distribution was obtained possibly because this acceleration effect could be negligible in high ratios of NCA/initiator. For block copolymer 5-10, the difference in the Asp unit number

298

Bioconjugate Chem., Vol. 3, No. 4, 1992

Table II.

Yokoyama et al.

Synthesis of PEO-P(Asp) feed composition

PEO-PBLA

run

1-10° 1-20 1-40 1-80 2-10 2-20 2-40 2-80 5-10 5-20 5-40 5-80 12-20 12-40 12-80

(g)

0.5

N NaOH solution (mL)

reaction time (min)

yield (g) [%]b

Asp units0

elution volume1* (mL)

53 26 54 120 50 20 70 180 40 22 80 25

1.21 [98.0] nde [nd]

9.5 20.5 41.6 83.8 8.8 18.8 39.5

14.6,15.9

35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 35.0 17.5 29.2 35.1

1.76 1.46 1.35 1.27 2.42 1.80 1.51 1.36 4.44 2.72 1.96 1.59 2.40 2.54 2.15

0.93 0.79 1.75 1.34

[113] [105] [92.4] [105] 1.11 [113]

14.6 14.1 13.4

14.2,15.6 14.1

13.5

/

3.10 [79.1] 2.16 [98.7] 1.53 [107] 1.22 [115] 2.02 [94.5]

12 12

2.10 [101]

20

1.63 [100]

8.1 17.6

15.1 14.7 13.9 13.3 14.1 13.7 13.2

36.0 75.2 17.9 35.8 70.8

Abbreviation used was based on molecular weight of the PEO chain and polymerization degree of BLA units, in the same way as in Table 1.6 Percent yield was calculated with the theoretical weight of PEO-P(Asp) transformed from PEO-PBLA as 100 %.c Determined by *H NMR d using methylene proton (3.7 ppm) of the PEO chain and methylene proton (2.8 ppm) of the P(Asp) chain. Gel-filtration chromatography with a Shodex OHpak KB-802.5 column and a KB-803 column in 0.1 M phosphate-buffered solution (pH 7.4).e nd: not determined. /The reaction mixture remained heterogeneous after 3 h of stirring. 0

Table III.

Introduction of ADR to PEO-P(Asp) reaction0 condition

PEO-P(Asp)

DMF (mL)

dialysis

1-10

25.0 12.5 25.0 25.0 12.5 12.5 6.3 25.0 25.0 12.5 6.3 25.0 25.0 12.5 25.0 25.0 25.0 12.5 12.5

2.0 8.0 2.0 8.0 4.0 8.0 16.0 2.0 8.0 16.0 16.0 2.0 16.0 16.0 2.0 4.0 8.0 4.0 8.0

precipitation precipitation precipitation precipitation precipitation

1-20

1-40

1-80

2-10

reaction0 condition

status6 after

ADR (mg)

PEO-P(Asp) 2-20 2-40

opaque homogenous

5-10 5-20 5-40 5-80

precipitation precipitation precipitation homogenous

precipitation precipitation precipitation precipitation

12-20 12-40 12-80

opaque homogenous homogenous homogenous

ADR (mg)

DMF (mL)

25.0 25.0 25.0 25.0 12.5 12.5 25.0 25.0 25.0 25.0 25.0 12.5 12.5 25.0 25.0 25.0 12.5

2.0 2.0 4.0 8.0 4.0 8.0 2.0 2.0 2.0 2.0 4.0 2.0 4.0 2.0 2.0 2.0 4.0

status dialysis

precipitation precipitation precipitation opaque homogenous homogenous homogenous homogenous opaque

precipitation opaque homogenous homogenous homogenous homogenous homogenous homogenous

0 Reaction conditions: triethylamine, 1.3 equiv to ADR; EDC, 13 mL + 13 fiL; PEO-P(Asp), quantity containing 9.1 X 10“4 mol of Asp unit; distilled water, 200 fiL; reaction time, 4 h (0 °C) + 15 h (room temperature). 25.0 mg of ADR-HC1 corresponds to a mol equiv (9.1 X 10“4 mol) of Asp units to PEO-P(Asp). 6 Status is shown with three words: precipitation, opaque, and homogenous.

Figure

3. (- -), 2-20

Gel-filtration chromatograms of PEO-P(Asp) 2-10

(---), and 2-40 (—): Column, Shodex OHpak KB-

802.5 + KB-803; eluent, 0.1 M phosphate-buffered solution (pH 7.4); flow rate, 1.0 mL/min; temperature, 40 °C; detection,

refractive index.

expected to become negligible in its molecular weight distribution due to its relatively long PEO chain. Binding of ADR to PEO-P(Asp). To obtain a watersoluble conjugate PEO-P(Asp(ADR)), several reaction conditions were investigated in the covalent attachment was

of ADR to PEO-P(Asp) by changing quantities of ADR and DMF as summarized in Table III. The faculty of obtaining a water-soluble conjugate was revealed to be primarily dependent on the composition of PEO-P(Asp) as compared in the reaction conditions as follows: 25.0 mg of ADR-HC1, 7.8 fiL of triethylamine, PEO-P(Asp) containing 1 mol equiv of aspartic acid (Asp) units to ADR, 200 fiL of distilled water, and 13 /^L + 13 /uL of EDC. Experiments carried out with high PEO content (e.g., 1220) brought about homogeneous solutions after overnight dialysis against 0.1 M sodium acetate buffer solution (pH 4.5), while runs with low PEO content such as 1-20 resulted in precipitation. The precipitation behavior, however, was not only dependent on the composition but also on lengths of the polymer chains. Run 12-80, which brought about

homogeneous conjugate, contains smaller weight fraction (59.6%) of the PEO chain than run 2-10 (66.5%) having resulted in precipitation in these conditions. This fact suggests that longer PEO chains were able to inhibit precipitate formation more efficiently than shorter PEO chains. For runs that resulted in precipitation under these conditions, homogeneous solutions were obtained by decreasing the ADR molar ratio and increasing the a

Btoconjugato Chem., Vol. 3, No. 4, 1992

Preparation of Micelle-Forming Polymer-Drug Conjugates

Table IV.

299

Synthesis of PEO-P(Asp(ADR)) ADR-HC1 (mg)/

run0

DMF (mL)

PEO-P(Asp) (mg)/ H2O (mL)

1-40 2-10 2-40 5-10 5-80 12-20 12-40 12-80

500/1280 700/224 700/224 600/48 600/96 600/48 600/48 600/48

480/16.0 829/11.2 400/11.2 763/4.8 375/9.6 810/4.8 465/4.8 295/4.8

ultrafiltration6 A B B B A A C

A

conjugated ADR' (mg) 1% yield] 110.9 509.6 404.0 428.7 398.4 369.2 454.6 252.9

[22.2] [72.8] [57.7] [71.5] [66.4] [61.5] [77.4] [42.2]

substitution*4

ratio (%) 12

44 30 73 46 104 78 84

diameter' (nm)

nd/ nd/ 21,123 24,131 36,118 40 58

14,91

molecular weight of PEO chain and polymerization degree of Asp units, in the same way as with Table I. 6 Ultrafiltration procedure is abbreviated as follows: (A) with a Minitan system (Millipore) equipped with a membrane of molecular weight cutoff 100 000, followed by Amicon stirring cells equipped with a PM-30 membrane (Amicon; molecular weight cutoff 30 000), (B) with a Minitan system equipped with a membrane of molecular weight cutoff 10 000, followed by Amicon stirred cells equipped with a UK-10 membrane (Advantec, Tokyo, Japan; molecular weight cutoff 10 000), (C) with Amicon stirred cells equipped with a PM-30 membrane. c Measured by absorbance at 485 nm in DMF. d Percent molar substitution of ADR with respect to Asp residues of PEO-P(Asp). Calculation method is described in the text.' Weight average diameter of each peak is shown. / nd: not detected; diameter was not detected above 10 nm. “

Abbreviation used based

on

quantity of DMF. For 2-10 with ADR ratio of 1, solution status after the dialysis was changed from precipitation to homogeneity by increasing the quantity of DMF from 2.0 to 8.0 mL. Decreasing the molar ratio of ADR was also effective to prevent precipitation from occurring in the introduction reaction as shown by several examples such 5-80. According to the study of the reaction conditions in Table III, ADR was introduced to eight kinds of compositions of PEO-P(Asp) as summarized in Table IV. For all the eight runs, the reaction had proceeded homogeneously, and the reaction mixtures were homogeneous after the dialysis against 0.1 M sodium acetate buffer (pH 4.5) for 6 h. Then, the reaction mixtures were purified by repeated ultrafiltration with distilled water until the ADR concentration of the filtrate was reduced to less than V1000 of the ADR concentration of the residual solution on ultrafiltration membranes. The amounts of the conjugated ADR in the polymeric drugs were determined by measuring the absorbance at 485 nm in DMF. The substitution ratios of the Asp units with ADR were calculated after lyophilization. The weight of the PEO-P(Asp) fraction in a dried PEO-P(Asp(ADR)) sample was obtained by subtracting the conjugated ADR weight from the total weight of the lyophilized sample. The substitution ratios were calculated on the assumption that the compositions of PEOP(Asp) did not change during the conjugation reaction and the following purification procedures. These ratios are expressed as mole percents with respect to the aspartic acid (Asp) residues of the PEG-P(Asp) chain as summarized in Table IV. For all the runs except 1-40 and 2-40, higher ADR contents were obtained than the previously reported value (30 mol %) for 5-20 (14). A decrease in DMF quantity was found to contribute to obtaining conjugates with high ADR contents, since DMF quantity in the previous paper corresponded to 9.4 times that for 5-20 in Table III. For 12-20, a substitution ratio resulted in a value over 100%. It is considered that this value was reflected by an error in the ^-NMR measurement of PEO-P(Asp). Micelle formation was confirmed by gel-filtration chromatography for all eight samples of PEO-P(Asp(ADR)) as typified by charts of 1-40, 2-10, 5-10, and 12-20 in Figure 4. A peak at the gel-exclusion volume (4.2 mL), which corresponds to a molecular weight of over 300 000 on the pullulan standard, was observed for all runs. This peak indicated the elution of the conjugates as polymeric micelles because the molecular weights of these conjugates were much smaller than 300 000. The elution profile was, however, dependent on the composition. For PEO-P(Asp(ADR)) 1-40, a small fraction appeared at the gel-exclusion volume, followed by a larger peak at 6.9 mL, which was as

elution volume

elution volume

Figure 4. Gel-filtration chromatograms of PEO-P(AspCADR))

(a) 1-40, (b) 2-10, (c) 5-10, (d) 12-20: Column, Asahipak GS-520 H; eluent, 0.1 M phosphate-buffered solution (pH 7.4, containing 0.3 M NaCl); flow rate, 1.0 mL/min; temperature, 40 0 C; detection, absorption at 485 nm.

considered a polymer fraction not forming a micellar structure. For PEO-P(Asp(ADR)) 2-10, a sharp peak at the gel-exclusion volume was accompanied by a small shoulder around 7 mL. PEO-P(Asp(ADR)) 5-10 gave a broader peak than the others. The other compositions were observed to show distinct micelle formation by a sharp peak at the gel-exclusion volume as typified by a chart of 12-20. From these facts, it is considered that longer chains are favorable in both the segments to form distinct micellar structures. Micelle size was measured by dynamic laser light scattering. For 1-40 and 2-10, micelles of over 10 nm in diameter were not observed, possibly due to short chain lengths of the PEO segment. Unimodal diameter distribution was obtained only for 12-20 and 12-40, while the other composition brought about bimodal distribution as summarized in Table IV and shown in Figure 5. These micelles were revealed to be formed by noncovalent hydrophobic interaction since all the micelle peaks in the laser scattering measurements disappeared by an addition of 1 wt % of sodium dodecyl sulfate. Although diameters of the micelles were not determined only by the chain lengths of the conjugates, possibly because of variety in

300

Bioconjugate Chem., Vol. 3, No. 4, 1992

Yokoyama at al.

form micellar structures by changing the ADR ratio to the Asp residues. Efficient targeting of the micelles can be optimized with this wide range of chain lengths of both

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diameter diameter Figure 5. Diameter distribution of PEO-P( Asp (ADR)): (a) 1220, (b) 12-40, (c) 12-80, (d) 5-80, measured with a dynamic laser light scattering spectrometer equipped with an argon laser beam at a concentration of 20 fig ADR-HC1 equiv/mL in phosphatebuffered saline (Na/Na, pH 7.4, 0.155 M) at 25 °C.

the aggregation numbers of the micelles, it was found that micelle-forming polymeric drugs with a range from approximately 10 to 100 nm in diameter were obtained with various compositions in the block copolymer. It was found that all the runs in Table IV maintained water solubility irrespective of high loading of hydrophobic ADR residues to the block copolymer even after being concentrated to 20 mg ADR equiv/mL, and that they were able to be injected into tail vein of mice without sudden deaths caused by precipitation in the blood vessels. The results of in vivo anticancer activity of these conjugates will be reported elsewhere. DISCUSSION

The conjugates of ADR and PEO-P(Asp) were successfully synthesized without precipitation for the four lengths in both the segments by changing the ratio of ADR to the Asp residues and the quantity of DMF. Although water-soluble conjugates were not obtained for four copolymers, 1-10, 1-80, 2-20, and 5-40, it is considered that ADR could be bound to PEO-P(Asp) block copolymer without precipitation for all the samples except 1-80 because ADR could be successfully bound to the block copolymer with a shorter P(Asp) chain by the reaction conditions which brought about a homogeneous conjugate for the block copolymer with a longer P(Asp) chain. For example, the conjugate 1-10 may be synthesized by the reaction conditions (6.3 mg of ADR and 16.0 mL of DMF) which brought about the water-soluble conjugate for 120. As a result, micelle-forming conjugates were obtained from PEO-P(Asp) containing from 17 wt % (1-40) to 85 wt % (12-20) of the PEO chain. Generally, the range of composition for block copolymers to form micellar structures is not so large. For one example, PEO-poly(styrene) block copolymer was reported to form micellar structures in distilled water of PEO weight content only from 61 % to 80% (24). The wider range of PEO weight content for PEO-P(Asp(ADR)) than that for the PEO-poly(styrene) block copolymer is considered to result from the binding method of ADR to PEO-P(Asp) because the hydrophobichydrophilic balance of the conjugate can be adjusted to

approximately 10 to 100 nm were obtained with PEOP(Asp(ADR)). This diameter range is very interesting for drug targeting because it was reported that some specially designed liposomes (7,8) with diameter of about 100 nm circulated stably in blood for long time periods and that nanospheres (25) (microspheres below 1 fim in diameter) could be utilized for drug targeting by intravenous injection. Furthermore, the architecture of polymeric micelles is very unique among all the drug-delivery systems because of their hydrated soft shells, and this architecture cannot be obtained in the other types of carrier systems. Therefore, polymeric micelles are expected to express their targeting behavior (e.g. biodistribution, interaction with cells, and permeation into tissues) differently from the conventional systems. The diameter of the polymeric micelles could be primarily controlled by the chain lengths mainly of PEO. Fine control of the micelle size, however, was not achieved, possibly because of various aggregation numbers of the micelles. Detailed physicochemical studies to describe the relationship between the composition of PEO-P(Asp(ADR)) and micelle size and stability are needed to design the polymeric micelle carrier for more efficient drug targeting. High water solubility of the conjugates in Table IV proved an outstanding property of a micellar form for carrying hydrophobic drugs while maintaining high water solubility. For conventional polymeric carriers, several groups reported maximum mole percent substitution of side carboxyl groups of polymer chains by ADR or daunomycin (an ADR analogue): 5 mol % (10) and 10 mol % (26) for poly(glutamic acid), 8 mol % (27) for pyran copolymer, and 3 mol % (9) for poly[AT-(2-hydroxypropyl)methacrylamide]. For PEO-P(Asp(ADR)), ADR was bound to the side carboxyl groups almost quantitatively as shown by 12-20. As compared in ADR weight percent in the conjugates, PEO-P(Asp(ADR)) also afforded much larger values (60% for 12-80) than those of the conventional polymer drugs: 17 wt % (10) and 26 wt % (26) for poly(glutamic acid), 35 wt % (27) for pyran copolymer, and 10 wt % (9) for poly[iV-(2-hydroxypropyl)methacrylamide]. Furthermore, PEO-P(Asp(ADR)) containing many ADR residues did not cause a precipitate by the concentrating procedure or temperature change from 0 °C to room temperature, while a conventional polymer drug was reported to precipitate by temperature change at around room temperature (9). Such stable water solubility is expected to contribute to safe injection of the conjugate into the blood stream by inhibiting precipitation in the blood vessels. This excellent water solubility is considered to result from the core-shell structure of the micelle in which the outer shell can inhibit aggregation of the hydrophobic cores very efficiently. All these results pointed out that micellar forms are very suitable to design a hydrophobic drug-carrying system. Although detailed physicochemical characterization of the micelles is indispensable to establish this new carrier system, a unique delivery system may be constructed with polymeric micelles. For polymeric micelles, factors to determine their pharmacokinetic behavior and biodistribution are the chemical characters of the outer shell and the size and stability of the micelles. These three factors are independent of drugs bound to the inner core. Therefore, drug delivery with polymeric micelles can be controlled drug-independently, while delivery of the conventional polymeric drug was affected by bound drug

Bioconjugate Chem., Vol. 3, No. 4, 1992

Preparation of Micelle-Forming Polymer-Drug Conjugates

because the bound drug faces outside to interact with biocomponents. If a polymeric micelle is analyzed pharmacokinetically, many kinds of drugs can be applied to this

polymeric micelle system by only adjusting the quantity of bound drug to correct the hydrophobic-hydrophilic balance of the conjugate for micelle formation. Therefore, this polymeric micelle drug-carrying system can be applied to many kinds of hydrophobic drugs. This paper presented successful preparations of the block copolymer-drug conjugates PEO-P(Asp(ADR» of various chain lengths in both the segments, and showed excellent water solubility of these conjugate irrespective of the large amount of bound ADR. This paper also showed micelle formation of these conjugates with diameters ranging from approximately 10 to 100 nm. These micelles can pass through niters with submicron pores, and therefore can be easily sterilized by a simple filtration procedure. This is another preferable pharmaceutical property of the polymeric micelles. LITERATURE CITED (1) Uadia, P., Blair, A. H., and Ghose, T. (1984) Tumor and

tissue distribution of a methotrexate-anti-EL4 immunoglobulin conjugate in EL4 lymphoma-bearing mice. Cancer Res. 44, 4263-4266. (2) Koizumi, M., Endo, K., Kunimatsu, M., Sakahara, H., Nakashima, T., Watanabe, Y., Saga, T., Konishi, J., Yamamuro, T., Hosoi, S., Toyama, S., Arano, Y., and Yokoyama, A. (1988) 67Ga-labeled antibodies for immunoscintigraphy and evaluation of tumor targeting of drug-antibody conjugates in mice. Cancer Res. 48, 1189-1194. (3) Yang, H. M., and Reisfeld, R. (1988) Doxorubicin conjugated with a monoclonal antibody directed to a human melanomaassociated proteoglycan suppresses the growth of established tumor xenografts in nude mice. Proc. Natl. Acad. Sci. U.S.A. 85, 1189-1193. (4) Thfedrez, P., Saccavini, J.-C., Nolibfe, D., Simoen, J.-P., Guerreau, D., Gestin, J. F., Kremer, M., and Chatal, J. F. (1989) Biodistribution of indium-lll-labeled OC125 monoclonal antibody after intraperitoneal injection in nude mice intraperitoneal grafted with ovarian carcinoma. Cancer Res. 49,30813086. (5) Davis, S. S., and

Ilium, L. (1986) Colloidal delivery systems—Opportunities and challenges. Site-Specific Drug Delivery (E. Tomlinson, and S. S. Davis, Ed.) pp 93-110, John Wiley & Sons Ltd., Lancing, Sussex, UK. (6) Gregoriadis, G., Senior, J., Wolff, B., and Kirby, C. (1984) Fate of liposomes in vivo: control leading to targeting. Receptor-Mediated Targeting of Drugs (G. Gregoriadis, G. Poste, J. Senior, and A. Trouet, Ed.) pp 243-266, Plenum Press, New York. (7) Allen, T. M., and Chonn, A. (1987) Large unilamellar liposomes with low uptake into the reticuloendothelial system. FEES Lett. 223, 42-86. (8) Gabizon, A., and Papahadjopoulos, D. (1988) Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors. Proc. Natl. Acad. Sci. U.S.A. 85, 6949-6953. (9) Duncan, R., Kopeckova-Rejmanova, P., Strohalm, J., Hume, I. , Cable, H. C., Pohl, J., Lloyd, J. B., and Kopecek, J. (1987) Anticancer agents coupled to N-(2-hydroxypropyl)methacry-

lamide copolymers I. Evaluation of daunomycin and puromycin conjugates in vitro. Br. J. Cancer 55, 165-174. (10) Hoes, C. J. T., Potman, W., van Heeswijk, W. A. R., Mud, J. , de Grooth, B. G., Grave, J., and Feijen, J. (1985) Optimization of macromolecular prodrugs of the antitumor antibiotic adriamycin. J. Controlled Release 2, 205-213. (11) Endo, N., Umemoto, N., Kato, Y., Takeda, Y., and Hara, T. (1987) A novel covalent modification of antibodies at their amino groups with retention of antigen-binding activity. J. Immunol. Methods 104, 253-258.

301

(12) Zunino, F., Pratesi, G., and Micheloni, A. (1989) Poly(car-

boxylic acid) polymers as carriers for anthracyclines. J. Controlled Release 10,65-73; Description was made in Table

II.

(13) Yokoyama, M., Inoue, S., Kataoka, K., Yui, N., Okano, T. (1989) Molecular design for missile drug: Synthesis of adri-

amycin conjugated with IgG using polyethylene glycol)-poly(aspartic acid) block copolymer as intermediate carrier. Makromol. Chem. 190, 2041-2054. (14) Yokoyama, M., Miyauchi, M., Yamada, N., Okano, T., Sakurai, Y., Kataoka, K., and Inoue, S. (1990) Characterization and anticancer activity of the micelle-forming polymeric anticancer drug adriamycin-conjugated poly (ethylene glycol)-poly(aspartic acid) block copolymer. Cancer Res. 50,1693-1700. (15) Yokoyama, M., Miyauchi, M., Yamada, N., Okano, T., Sakurai, Y., Kataoka, K., and Inoue, S. (1990) Polymer micelles as novel carrier: Adriamycin-conjugated polyethylene glycol)poly(aspartic acid) block copolymer. J. Controlled Release 11, 269-278. (16) Bader, H., Ringsdorf, H., and Schmidt, B. (1984) Watersoluble polymers in medicine. Angew. Chem. 123/124, 457485. (17) Pratten, M. K., Lloyd, J. B., Horpel, G., and Ringsdorf, H. (1985) Micelle-forming block copolymers: Pinocytosis by mac-

rophages and interaction with model membranes. Makromol. Chem. 186, 725-733. (18) Kabanov, A. V., Chekhonin, V. P., Alakhov, V. Yu., Batrakova, E. V., Lebedev, A. S., Melik-Nubarov, N. S., Arzhakov, S. A., Levashov, A. V., Morozov, G. V., Severin, E. S., and Kabanov, V. A. (1989) The neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles; Micelles as microcontainers for drug targeting. FEBS Lett. 258, 343-345. (19) Yokoyama, M., Okano, T., Sakurai, Y., Ekimoto, H., Shibazaki, C., and Kataoka, K. (1991) Toxicity and antitumor activity against solid tumors of micelle-forming polymeric anticancer drug and its extremely long circulation in blood. Cancer Res. 51, 3229-3236. (20) Yokoyama, M., Inoue, S., Kataoka, K., Yui, N., and Sakurai, Y. (1987) Preparation of adriamycin-conjugated polyethylene glycol)-poly(aspartic acid) block copolymer. Makromol. Chem. Rapid Commun. 8, 431-435. (21) Saudek, V., Picova, H., and Drobnik, J. (1981) NMR study of poly (aspartic acid). II. a- and /3-peptide bonds in poly(aspartic acid) prepared by common methods. Biopolymers 20, 1615-1623. (22) Sekiguchi,H. (1981) Mechanism of N-carboxy-a-amino acid anhydride (NCA) polymerization. Pure Appl. Chem. 53,1689.

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A study of the kinetics of the primary amine-initiated polymerization of N-carboxy-anhydrides with special reference to configurational and stereochemical effects. J. Am. Chem. Soc. 79, 3961-3972.

(24) Wilhelm, M., Zhao, C.-L., Wang, Y., Xu, R., Winnik, M. A., Mura, J.-L., Riess, G., and Croucher, M. D. (1991) Polystyrene-

ethylene oxide) block copolymer micelle formation in water: a fluorescence probe study. Macromolecules 24,1033-1040. (25) Douglas, S. J., Ilium, E., and Davis, S. S. (1986) Poly(butyl 2-cyanoacrylate) nanoparticles with differing surface charges. J. Controlled Release 3, 15-23. (26) Tsukada, Y., Kato, Y., Umemoto, N., Takeda, Y., Hara, T., and Hirai, H. (1984) An anti-a-fetoprotein antibody-daunorubicin conjugate with a novel poly-L-glutamic acid derivative as intermediate drug carrier. J. Natl. Cancer. Inst. 73, 721729.

(27) Hirano, T., Ohashi, S., Morimoto, S., Tsukada, K., Kobayashi, T., and Tsukagoshi, S. (1986) Synthesis of antitumor-

active conjugates of adriamycin or daunomycin with the copolymer of divinyl ether maleic anhydride. Makromol. Chem. 187, 2815-2824.

Registry No.

3, 141982-53-8.