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Synthesis of Starlike N-(2-Hydroxypropyl)methacrylamide Copolymers: Potential Drug Carriers† Dong Wang,‡ Pavla Kopecˇ kova´ ,‡ Tamara Minko,‡ Vajira Nanayakkara,§ and Jindrˇich Kopecˇ ek*,‡,| Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, Mass Spectrometry Facility, and Department of Bioengineering, University of Utah, Salt Lake City, Utah 84112 Received March 14, 2000; Revised Manuscript Received May 29, 2000
Starlike HPMA copolymers were synthesized by conjugating semitelechelic poly[N-(2-hydroxypropyl)methacrylamide] macromolecules (ST-PHPMA, arm) with PAMAM dendrimers (core: G2, G3, G4). STPHPMA was synthesized by chain transfer free radical polymerization, and the terminal -COOH was activated with N-hydroxysuccinimide. Doxorubicin (DOX) was introduced into the starlike HPMA copolymer to evaluate its potential as a drug delivery system. The polymers were characterized with SEC, NMR, and UV. Cytotoxicity of the DOX containing starlike HPMA copolymer was determined on an A2780 human ovarian carcinoma cell line and compared with DOX-containing linear HPMA copolymers. The rate of in vitro DOX release from polymer-DOX conjugates in the presence of cathepsin B (CP-B, lysosomal cysteine proteinase) was determined and correlated with cytotoxicity results. Introduction Starlike polymers are a group of spherical polymers containing more than two arms attached onto a common core. Many starlike designs have been proposed,1-6 possessing unique properties resulting from their three-dimensional structures. N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers have been designed, synthesized, and evaluated as anticancer drug carriers. They are hydrophilic, nonimmunogenic, and do not possess nonspecific toxicity (reviewed in ref 7). Drug containing monomers can be easily incorporated by copolymerization with HPMA. In Phase I clinical trials, the HPMA copolymer-doxorubicin (DOX) conjugate (PK1) demonstrated reduced dose-limiting toxicity (as compared to free DOX), while maintaining antitumor activity.8 HPMA copolymer-DOX conjugates appear to overcome the action of ATP driven efflux pumps in multidrug resistant (MDR) cells,9 resulting in better efficacy in treating MDR solid tumors when compared to free DOX.10 It appears that starlike polymers based on HPMA copolymers might have potential as new drug delivery systems. Their unique structures may have some interesting properties that are different from their linear counterparts such as a lower viscosity in solution as well as the possibility of attaching several drugs to the same carrier. Pioneering work with a starlike HPMA copolymer containing DOX has been done by Ulbrich’s group; they used an antibody as the core. The targeting activity of the conjugated antibody was reported to be only slightly affected.11 * To whom correspondence should be addressed. Telephone: (801) 5814532. Fax: (801) 581-3674. E-mail:
[email protected]. † See Experimental Section for List of Abbreviations. ‡ Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah. § Mass Spectrometry Facility, University of Utah. | Department of Bioengineering, University of Utah.
In the present study, we report the synthesis of starlike HPMA copolymers using Starburst PAMAM dendrimers (Scheme 1; ref 12) as the core. The starlike polymers were characterized by SEC, NMR, and UV. DOX was introduced into the starlike HPMA copolymer to verify its potential as a new drug delivery system. Cytotoxicity of DOX bound to the starlike polymer was determined on the A2780 human ovarian carcinoma cell line and compared with DOXcontaining linear HPMA copolymer conjugates. The in vitro release of DOX from HPMA copolymer-DOX conjugates in the presence of cathepsin B was determined and correlated with cytotoxicity data. Experimental Section Abbreviations: ACA, 4,4′-azobis(4-cyanopentanoic acid); AIBN, 2,2′-azobis(isobutyronitrile); Bz-Phe-Val-Arg-NAp, N-benzyol-PheVal-Arg-p-nitroanilide; CP-B, bovine spleen cathepsin B; dn/dc, refractive index increment; DOX, doxorubicin; DCC, dicyclohexylcarbodiimide; DCU, dicyclohexyl urea; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DPBS, Dulbecco’s phosphate buffered saline; EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatography; Gly-NAp, glycine p-nitroanilide; Gx, generation x of PAMAM dendrimers; HOSu, N-hydroxysuccinimide; HPMA, N-(2-hydroxypropyl)methacrylamide; MAGFLG-DOX, N-methacryloylglycylphenylanalanylleucylglycyl doxorubicin; MPA, 3-mercaptopropionic acid; MALDI-TOF MS, matrix-assisted laser desorption/ionization time-of-flight mass sepectometry; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; Mn, number-average molecular weight; Mw, weight-average molecular weight; MWD, molecular weight distribution; P(GFLG)-DOX, HPMA copolymer-DOX conjugate containing GFLG side chains terminating in DOX (see Scheme 2); PHPMA, poly[N-(2-hydroxypropyl)methacrylamide]; SEC, size exclusion chromatography; ST, semitelechelic; ST-PHPMA, semitelechelic PHPMA; ST-PHPMA-COOH, semitelechelic poly[N(2-hydroxypropyl)methacrylamide] having a -COOH terminus;
10.1021/bm0000236 CCC: $19.00 © 2000 American Chemical Society Published on Web 07/07/2000
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Scheme 1. Synthesis of Starlike PHPMA
Scheme 2. Structure of HPMA Copolymer-DOX Conjugates: A, P(GFLG)-DOX; B, ST-PHPMA-DOX-COOH
ST-PHPMA-COOSu, ST-PHPMA-COOH in which the -COOH had been converted to a N-hydroxysuccinimide ester; ST-PHPMADOX-COOH, semitelechelic poly[N-(2-hydroxypropyl)methacrylamide-co-N-methacryloylglycylphenylanalanylleucylglycyl doxorubicin] having a -COOH terminus; ST-PHPMA-DOXCOOSu, ST-PHPMA-DOX-COOH in which the -COOH had been converted to a N-hydroxysuccinimide ester; Sx, starlike PHPMA using generation x of PAMAM dendrimers as the core (see Scheme 1 for structure); S4-DOX, starlike HPMA copolymer using ST-PHPMA-DOX-COOH as the arms and PAMAM dendrimer (G4) as the core. Chemicals. Starburst (PAMAM) dendrimer (ethylenediaminetetrapropionic acid based dendrimer; see Scheme 1 for chemical structure;12 G2, G3, and G4), MPA, DCC, HOSu were purchased from Aldrich, Milwaukee, WI. MTT and ACA were purchased from Fluka, Ronkonkoma, NY. Gly-NAp, RPMI 1640 media, penicillin, CP-B and Bz-Phe-Val-Arg-NAp were purchased from Sigma, St. Louis, MO. Fetal bovine serum and insulin were purchased from
Hyclone, Ogden, UT. PD-10 columns, Sephadex LH-20 beads and Sephadex G-25 beads were purchased from Pharmacia Biotech, Piscataway, NJ. DOX was a kind gift from Dr. A. Suarato, Pharmacia-Upjohn, Milano, Italy. HPMA,13 MA-GFLG-DOX,14 and P(GFLG)-DOX9,15 were prepared as previously described. All the other solvents and reagents were reagent grade or better and used without purification. Cell Line. The A2780 human ovarian carcinoma cell line was obtained from Dr. T. C. Hamilton (Fox Chase Cancer Center, PA). Cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum, 10 µg/mL insulin, and 100 U/mL penicillin. Cells were grown at 37 °C in a humidified atmosphere of 5% (v/v) CO2. All experiments were performed on cells in the exponential growth phase. Methods. The polymers were characterized by SEC using an FPLC (Pharmacia) equipped with UV, RI, and laser light scattering (MINI DAWN, Wyatt Technology, Santa Barbara, CA) detectors. SEC measurements were carried out on Superose 6, Superose 12,
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Starlike HPMA Copolymers or Superdex 75 columns (analytical) with PBS buffer as the eluent. The average molecular weights of the polymers were calculated with ASTRA Chromatography Software (Wyatt Technology) based on the data from laser light scattering. The dn/dc of each of the polymers were determined in PBS buffer on an OPTILAB DSP Interferometric refractometer of Wyatt Technology. End group analysis of semitelechelic polymers was obtained by MALDI-TOF MS (Voyager-DE STR Biospectrometry Workstation, PerSeptive Biosystems, Inc., Framingham, MA). It was equipped with a nitrogen laser with a wavelength of 337 nm. The instrument was operated at a 20 kV accelerating voltage with the use of delayed extraction and the low mass gate. 2-(4-Hydroxyphenylazo) benzoic acid was used as the matrix for the polymers. Sodium chloride was used as the ionic reagent to facilitate the ionization process in the analysis of polymers. All spectra were obtained in a linear mode with the instrument calibrated externally using peptides or proteins in the same solution and matrixes as used for sample analysis. 1H NMR was performed on a Varian Unity 500 MHz NMR spectrometer. Solvent peak was used as reference (d6-DMSO, 2.49 ppm). UV spectra were obtained on a Lambda 6 UV spectrometer. Synthesis of ST-PHPMA-COOH. The synthesis was performed as previously described.16 Briefly, HPMA (2.0 g, 14.0 mmol), MPA (38 µL, 0.42 mmol) and ACA (168.2 mg, 0.6 mmol) were dissolved in 20 mL of methanol. The solution was purged with nitrogen, sealed in the ampule and polymerized at 50 °C. The polymer solution was concentrated and precipitated into diethyl ether. The polymer was purified three times by reprecipitation.16 Mn ) 4000; Mw ) 6800 as determined by SEC on a Superdex 75 column (dn/dc ) 0.170). The polymer was fractionated on a Superdex 75 preparative column using the FPLC system. Polymer fractions were collected, desalted by dialysis and lyophilized. The middle fraction (Mn ) 4800; Mw ) 5400) was selected for conjugation with dendrimers. Synthesis of ST-PHPMA-COOSu. ST-PHPMA-COOH (middle fraction, 1.3 g, 0.23 mmol) and HOSu (0.45 g, 3.9 mmol) were dissolved in 25 mL of DMF and cooled to 0 °C. DCC (0.81 g, 3.9 mmol) was dissolved in 5 mL of DMF and dropped slowly into the polymer solution. After addition, the solution was stirred at 4 °C for 48 h and then an additional 12 h at room temperature. Acetic acid (210 µL) was added to the solution and stirred for 2 h. DCU was filtered off, and the filtrate was precipitated into ether. The polymer was purified three times by reprecipitation.16 The conversion of -COOH to -COOSu was determined by model aminolysis with an excess of Gly-NAp as 81%. In this reaction, DCC and HOSu were used in large excess to prevent the side reaction between the -OH of HPMA and the -COOH terminal group. SEC results showed that there was no change in molecular weight of the polymers before and after esterification, which indicated the absence of the side reaction mentioned above. Synthesis of ST-PHPMA-DOX-COOH. HPMA (1.0 g, 7.0 mmol), MPA (18.3 µL, 0.21 mmol), ACA (84.1 mg, 0.3 mmol), and MA-GFLG-DOX (180 mg, 0.183 mmol) were dissolved in methanol (10 mL). The solution was purged with N2, sealed in an ampule, and polymerized at 50 °C. The polymer solution was precipitated into diethyl ether and dried under vacuum. The polymer was further purified on an Sephadex LH-20 column (in methanol) to eliminate free MA-GFLG-DOX. The purified polymer was fractionated by SEC on a Superdex 75 preparative column (PBS buffer containing 30% acetonitrile). The middle fraction was collected, desalted by dialysis and then lyophilized. The content of DOX in the copolymer was calculated from the UV spectrum
as 1.37 mol % (480 nm, ) 10500 in H2O). Mn ) 4700; Mw ) 5600. Synthesis of ST-PHPMA-DOX-COOSu. The synthesis was carried out in the dark using the same protocol as for the STPHPMA-COOSu as described above. Conversion of -COOH to -COOSu as determined by model aminolysis with an excess of Gly-NAp was 93%. The UV spectrum of the product indicated that no structural changes in the DOX moiety occurred during the reaction. Synthesis of Starlike HPMA Copolymer Using PAMAM Dendrimer as the Core. A typical synthesis of the starlike HPMA copolymers is described below. A PAMAM dendrimer solution (G4, 1.11 mg in methanol, 7.8 × 10-5 mmol of dendrimer or 0.005 mmol of -NH2) was dried, dissolved in DMSO (0.2 mL), and slowly dropped into the solution of ST-PHPMA-COOSu (250 mg, 0.05 mmol of -COOSu, 10 × molar excess over NH2 group, in 0.8 mL DMSO) with vigorous stirring. After 1 h, triethylamine (0.1 mmol) was added to the solution. The solution was stirred at room temperature for 3 days and then precipitated into acetone. Starlike HPMA copolymer was separated from the unreacted precursor by SEC (Superose 12 column, preparative), concentrated, dialyzed, and lyophilized. Mn ) 160 000; Mw ) 187 000 as determined by SEC on a Superose 12 column (dn/dc ) 0.164). 1H NMR (DMSO-d6): δ ) 2.1-2.7, 3.05, and 7.92 ppm (Hs of dendrimer); 0.81-1.01, 1.57, 2.9, 3.67, 4.7, and 7.17 ppm (Hs of PHPMA) (see Figure 2). The starlike HPMA copolymer containing DOX was synthesized using the same protocol but with ST-PHPMA-DOX-COOSu as the arm. The synthesis was carried out in the dark. The amount of unreacted amino group in the starlike polymers after conjugation was determined with a ninhydrin assay (glycine was used in the calibration). About 75-80% of the theoretical amino groups of the dendrimers could be detected with this assay (Table 2). However it was not applicable to starlike HPMA copolymers containing DOX because of the interference of DOX absorption and ninhydrin assay. Cytotoxicity Assay. The cytotoxicity of DOX bound to starlike and linear HPMA copolymers was assessed with a modified MTT assay.9 Briefly, cells were seeded into 96-well microtiter plates at the density of 10 000 cells per well. Twenty-four hours after plating, the medium was aspirated and 12 different concentrations of sterile drugs in fresh media were added. Control cells were treated with an equivalent volume of DPBS. Cells were cultured for 72 h with the drugs before the cell survival assay was performed. The medium was discarded and 100 µL of fresh media and 25 µL of a 5 mg/mL MTT solution in DPBS were added to each well. Plates were incubated under cell culture conditions for 3 h. Formazan crystals were dissolved overnight in 50% (v/v) DMF in water containing 20% (w/v) sodium dodecyl sulfate. The absorption of each sample was measured at 570 nm with a background correction at 630 nm. Cell Viability. The results of the cytotoxicity assay were used for the calculation of cell viability after incubation with drugs. The calculation can be described by the following equation:9
V ) (Y - Y0)/(Ym -Y0) ) (1 + C/C0)-1 Hence
Y ) Y0 + (Ym - Y0)/(1 + C/C0) where V is the cell viability, C is the drug concentration, C0 is the IC50 dose (the concentration of drug necessary to inhibit the growth of the cells by 50%), Y is the optical density in a well containing a particular drug concentration, C, Ym is the optical density which
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corresponds to 100% cell viability, and Y0 is the optical density which corresponds to 0% viability (dead cells). Ym was measured by extrapolation of the empirical curve to C ) 0 based on the results obtained with control cells grown under identical conditions. Two other parameters, Y0 and C0, were estimated using a nonlinear regression method and least-squares analysis. In vitro Release of DOX from HPMA Copolymer-DOX Conjugates. CP-B is a lysosomal enzyme involved in the intracellular digestion of proteins and HPMA-copolymer drug conjugates.17 In the present study, CP-B was used as a model enzyme to evaluate the in vitro release of DOX from HPMA copolymer-DOX conjugates. Conjugates (≈ 5 mg) were dissolved in the incubation mixture (37 °C, 5 min preincubation): CP-B stock solution (0.98 mL, 0.572 mg/mL in phosphate buffer, pH ) 5.5, 1 mM EDTA); cysteine solution (0.02 mL, 250 mM in phosphate buffer pH ) 5.5, 1 mM EDTA). At chosen time intervals, samples were withdrawn (0.1 mL), mixed with Na2CO3/NaHCO3 buffer (1 mL, 0.2 M, pH ) 9.8), and extracted with ethyl acetate (1.5 mL). The organic layer was separated and dried. The concentration of free DOX in ethyl acetate was determined with UV spectrophotometry (480 nm, ) 11000). The activity of the enzyme at different times of incubation was monitored using Bz-Phe-Val-Arg-NAp as the substrate. CP-B stock solution (0.025 mL), phosphate buffer (0.935 mL) and cysteine stock solution (0.02 mL) were mixed and incubated at 37 °C for 5 min. Bz-Phe-Val-Arg-NAp (0.02 mL, 0.05 M in DMSO) was mixed with the above solution and the UV absorption of released -NAp was monitored at 410 nm. The absorbency differences after 10 min of incubation at different time intervals were as follows: ∆A (1 cm, 410 nm) ) 2.03 (0 h), 1.92 (9 h), 1.51 (19 h), 1.18 (29 h), 0.72 (44 h).
Results and Discussion There are two major routes for the synthesis of starlike polymers: the divergent approach and the convergent approach.18 Monomers grow symmetrically from the core into the surrounding space to form a starlike polymer in the divergent approach, while in the convergent approach, tailormade polymer segments are attached onto the core by a coupling reaction between surface functional groups of the core and the end groups of the polymer (arm). There are several advantages to synthesizing a starlike drug delivery system with a convergent approach. First, the dose of the drugs can be easily varied over a wide range by modifying the drug content per macromolecule and by combining arms loaded with and without drugs onto the same core. Second, two different drugs may be incorporated into the same starlike polymer. It is well-known that combination therapy has been proven to be more effective than treatment with only one drug.19-21 However, the attachment of two drugs per one macromolecule usually impairs the solubility of the conjugate. The attachment of two arms containing different anticancer drugs to one PAMAM core has the potential to produce a soluble conjugate. For these reasons, the convergent approach was applied in the present study. PAMAM dendrimers, generations G2, G3, and G4 were used as the cores. They contain numerous amino groups at the core surface. ST-PHPMAs were used as arms. The terminal -COOH groups of ST-PHPMA were
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Figure 1. MALDI-TOF mass spectrum of ST-PHPMA-COOH using 4,4′-azobis(4-cyanopentanoic acid) as the initiator. Peak series “A” represents ST-PHPMA initiated by MPA free radicals and terminated by proton from MPA. Peak series “B” represents the STPHPMA initiated by the radical from ACA and terminated by proton from MPA.
converted into reactive HOSu ester groups and attached onto dendrimers via aminolysis of the -NH2 groups of dendrimers (Scheme 1). A starlike HPMA copolymer-DOX conjugate was synthesized by a similar procedure, using a STPHPMA-DOX arm (Scheme 2). The latter was prepared by radical chain transfer copolymerization of HPMA and MA-GFLG-DOX. Structure of Semitelechelic HPMA Copolymers. The synthesis of semitelechelic polymers containing a functional group at one end of the molecule is a prerequisite for their one point attachment to the surface of the dendrimer core. We have synthesized ST-PHPMA-COOH previously, using AIBN as the initiator and MPA as the chain transfer agent.16,22,23 However, free radicals formed by the decomposition of AIBN initiated a fraction of polymer chains; such chains were not terminated in a functional group. The amount of these polymer chains depended on the ratio between chain transfer agent and HPMA, the polymerization time, and concentration of AIBN.23 To eliminate the formation of nonfunctionalized ST-PHPMA, an azoinitiator containing -COOH groups, ACA, was used in the present study. We are aware that the use of functionalized initiators without chain transfer agents will result in a mixture of semitelechelic and telechelic macromolecules due to two mechanisms of termination of growing polymer chains (disproportionation and recombination).22 However, we hypothesized that the use of functionalized initiators in the presence of potent chain transfer agents, such as MPA, should result in semitelechelic polymers containing one of two (originating from MPA or ACA) functional -COOH containing chain ends. To verify the hypothesis, the end group structure of STPHPMAs was analyzed with MALDI-TOF MS. Although this technique may give incorrect data for average molecular weight, it offers an opportunity to directly determine the molar mass of individual polymer chains and, consequently, their exact composition.24-26 The MALDI-TOF mass spectrum of the ST-PHPMA-COOH (Figure 1) contains two peak series corresponding to macromolecules with different end group structures. The mass increment between peaks
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Figure 2. 1H NMR spectra of starlike HPMA copolymer (S4), dendrimer (G4), and PHPMA.
with identical end groups was 143.2, the mass of the HPMA monomer unit. Peak series “A” at m/z ) n × 143.2 + 23 (Na+) + 105.1 + 1.0 (n is the number of monomer units; mass of the MPA free radical is 105.1) corresponds to STPHPMA initiated by MPA free radicals and terminated by a proton from the MPA. The peak series “B” at m/z ) n × 143.2 + 23 (Na+) + 126.1 + 1.0 (molar mass of ACA free radical is 126.1) represents the ST-PHPMA initiated by the radical from ACA and terminated by a proton from the MPA. Peaks corresponding to the telechelic macromolecules were not observed, indicating that the recombination of radicals was indeed negligible under the experimental conditions used. Synthesis of Starlike HPMA Copolymers. Starlike HPMA copolymers were synthesized by aminolysis of -COOSu groups at ST-PHPMA termini with the surface -NH2 groups of dendrimers (Scheme 1). After the conjugation, the starlike copolymers were separated from unreacted precursor (used in 10 times excess) by SEC. In the 1H NMR spectrum of a starlike HPMA copolymer (Figure 2), peaks a (2.1-2.7 ppm) and peak b (3.05 ppm) are assigned to the proton of -CH2- in dendrimers. Peak d (7.92 ppm) is assigned to the proton of the amide in dendrimer. Peaks c (7.5-7.8 ppm) may be assigned to the proton of the amide bond formed between the dendrimer and the ST-PHPMA after conjugation. Their existence (especially peak c) indicates the attachment of the ST-PHPMA to the dendrimer core. Starlike HPMA copolymers were synthesized with three generations of dendrimers (G2, G3, G4). The SEC profiles of the starlike copolymers, dendrimers and ST-PHPMA are shown in Figure 3. The apparent average molecular weights of the polymers were calculated with ASTRA Chromatography Software (Wyatt Technology) based on the data from laser light scattering. The number of arms in the starlike polymers was estimated with the following equation: n ) (MS - MD)/MST n is the number of arms; MS, MD, and MST represent the apparent molecular weights of starlike PHPMA, dendrimer, and ST-PHPMA, respectively. The data (Table 1) revealed that all starlike PHPMA products possessed a narrow MWD (
