Biomacromolecules 2003, 4, 704-712
704
Water-Soluble Degradable Hyperbranched Polyesters: Novel Candidates for Drug Delivery? Chao Gao,†,‡ Yimin Xu,† Deyue Yan,*,† and Wei Chen† College of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240, P. R. China, and The Key Laboratory of Molecular Engineering of Polymers, Fudan University, Ministry of Education, P. R. China Received November 22, 2002; Revised Manuscript Received January 29, 2003
A novel approach to hyperbranched polymers is presented in this work. Hyperbranched polyesters with a large amount of terminal hydroxyl groups are prepared by a one-pot synthesis from commercially available AB-type and CDn-type monomers (n g 2). In this paper, Michael addition of diethanolamine (CD2) or N-methyl-D-glucamine (CD5) to methyl acrylate (AB) generates dominantly ADn-type intermediates. Further self-condensation of intermediates at higher temperature and in the presence of catalyst gives hyperbranched polyesters. Because of the tertiary amino groups in the backbone and the hydroxyl groups in the linear and terminal units, the resulting hyperbranched polyester is highly soluble in water. Furthermore, the hyperbranched polymer is degradable because of its ester units. So, the water-soluble hyperbranched polyesters might be applied as a novel material for drug delivery. Introduction Both dendrimers, and hyperbranched polymers are threedimensional highly branched macromolecules with numerous functional groups.1-9 Because of their unique features such as high solubility, low viscosity, and abundance of terminal groups, dendritic polymers are intriguing to chemists, biochemists, biologists, and biomedical experts.10 Dendrimers canbesynthesizedthroughdivergentorconvergentapproaches.11-17 Although its perfect monodisperse structure attracts much attention of researchers, the preparation of a dendrimer is generally costly and laborious because of numerous protection, deprotection, and purification steps.18 In contrast, polydisperse hyperbranched polymers can be obtained by one-step polycondensation of an ABn-type monomer.19-22 Depending on their special characteristics and properties aforementioned, dendritic polymers are good candidates as drug-delivery materials.23-26 The well-known dendrimers such as Tomalia-type polyamidoamine (PAMAM) dendrimer and Fre´chet-type dendrimer have been widely studied in the application of drug delivery.27,28 Because polyesters are highly degradable in water,29-34 much attention of drugdelivery systems is focused on the branched ones. First of all, drug-polymer conjugates require high water solubility; otherwise, problems might be caused after injecting them into the blood stream.23 Extensive works on dendritic polyesters have been published;35-45 however, as far as we know, water-soluble ones are rare. On the basis of the molecular design, this work presents a novel approach to water-soluble hyperbranched polyesters with high molecular weight. Through this approach, even water-soluble hyper* To whom correspondence should be addressed. E-mail: dyyan@ sjtu.edu.cn. Fax: +86-21-54741297. † Shanghai Jiao Tong University. ‡ Fudan University.
branched polyesters containing chiral glucamine units (similar to the structure of glucose) can be easily obtained.
Experimental Section Materials. Methyl acrylate (MA) was commercially purchased and purified by reduced-pressure distillation before use. Diethanolamine (DEOA), N-methyl-D-glucamine (NMGA), diethylamine, and 1,4-butanediol were purchased from Aldrich and used as received. Catalysts tetrabytyl titanate, Ti(C4H9O)4, and zinc acetate anhydrous, Zn(CH3CO2)2, and organic reagents and solvents such as benzoyl chloride, dimethyl sulfoxide (DMSO), chloroform, methanol, pyridine, and acetone were analytical pure reagents and used without purification. Characterization. Fourier-transform infrared (FT-IR) measurements were carried out on a Bruker Equinox 55 spectrometer.1H and 13C nuclear magnetic resonance (NMR) measurements of the resulting hyperbranched polymers were performed on a 500 MHz Bruker NMR spectrometer with DMSO-d6 as solvent. In situ 1H NMR measurements were carried out in the solution of CD3OD. The inverse-gated spectra were taken when the quantitative analysis of 13C NMR data was done because of the nuclear Overhauser effect. Mass spectra were obtained on a HP 1100 mass spectrograph detector (MSD). The conditions of spray chamber were given as follows: ionization mode, APCI; polarity, positive; fragmentor, 70 v; nebulizer pressure, 60 psig; drying gas flow, 7.0 mL/min; drying gas temperature, 325 °C. Differential scanning calorimetric characterization was conducted under nitrogen on a PE Pyris-1 DSC thermal analyzer. All samples were heated at 20 °C/min heating rate
10.1021/bm025738i CCC: $25.00 © 2003 American Chemical Society Published on Web 03/12/2003
Biomacromolecules, Vol. 4, No. 3, 2003 705
Water-Soluble Degradable Hyperbranched Polyesters
Table 1. Polymerization of Methyl Acrylate (MA) with Diethanolamine (DEOA) or N-methyl-D-glucamine (NMGA) code
CDn
catalysta
temp (°C)b
Mn1c
Mw/Mn1
ηinh (dL/g)
Mn2d
DB (%)
PAE-01 PAE-02 PAE-03 PAE-04 PAE-05 PAE-06 PAE-07 PAE-08 PAE-09 PAE-10 PAE-11
DEOA DEOA DEOA DEOA DEOA DEOA DEOA NMGA NMGA NMGA NMGA
Zn(OAc)2 Zn(OAc)2 Ti(C4H9O)4 Ti(C4H9O)4 Ti(C4H9O)4 Ti(C4H9O)4 Ti(C4H9O)4 Zn(OAc)2 Zn(OAc)2 Ti(C4H9O)4 Ti(C4H9O)4
150 140 150 165 140 135 120 145 135 145 135
80 650 50 160 268 340 gel 139 530 86 030 14 740 51 860 38 010 66 320 52 290
1.14 1.40 2.41
0.52 0.25 1.78
115 300 70 850 338 200
53.4 52.5
1.04 1.14 1.52 1.35 1.12 1.13 1.08
0.92 0.55 0.11 0.25 0.18 0.34 0.26
201 430
55.8
a Amount of catalyst was 0.5 g per mole of CD -type monomer. b The temperature within the last 2 h. The initial reaction temperatures were 60 °C for n 1 h, 100 °C for 2 h, and then 120 °C for 2 h. c The number-average molecular weight of the hyperbranched polymer with hydroxyl end groups. d The number-average molecular weight of the end-capped hyperbranched polymer.
from 35 to 200 °C for the first scan, then cooled at 20 °C/ min to -80 °C, and immediately heated with 20 °C/min from -80 to 160 °C for the second scan. Thermogravimetric analysis (TGA) was performed under nitrogen on a PE Pyris-7 thermal analyzer; all samples were heated with a heating rate of 20 °C/min from 25 to 650 °C. The molecular weight and its distribution of the hyperbranched polymer with hydroxyl end groups were obtained on the HP 1100 gel permeation chromatograph (GPC) with water as solvent and PEO as standards, and the column used was G6000 PW (XL). Optilab Dawn EOS multiangle laser light-scattering (MALLS) apparatus was used to measure the molecular weight of the end-capped hyperbranched polymer with polystyrene as standards and tetrahydrofuran (THF) as solvent. The inherent viscosity (ηinh) of the resulting polymer was measured at a concentration of 0.5 g/dL in DMSO at 30 °C. Synthesis of Hyperbranched Poly(MA-DEOA). A typical example (PAE-01 in Table 1) is given as follows. In a flask were placed 0.1 mol of DEOA, 0.105 mol of MA, and 20 mL of methanol. The mixture was kept at room temperature (about 25 °C) for 48 h with stirring. Then the flask was connected with a revolving-distillation apparatus. Under reduced pressure, the residual MA and methanol in the revolving-distillation apparatus were removed from the reaction system. Then 0.05 g of Zn(CH3CO2)2 was added into the flask. Under vigorous revolving and vacuum distillation, the mixture was kept at 60 °C for 1 h, 100 °C for 2 h, 120 °C for 2 h, and 150 °C for 2 h. The raw product was dissolved in 50 mL of DMSO and then poured into 1000 mL of acetone. The precipitate was collected and purified by reprecipitation from DMSO solution into acetone. A yellow rubber-like solid, 12.5 g (yield 78.6%), was obtained. IR (KBr): 3396.6 cm-1 (-OH), 1730.6 cm-1 (CdO). 1H NMR (DMSO-d6, ppm): δ 4.08 (-OH), 3.45 (CH2O), 2.45 (CH2N), 1.6 (CH2CdO). 13C NMR (DMSO-d6, ppm): δ 172.345, 171.38, 171.02, 69.23, 67.18, 66.18, 62.53, 59.55, 57.24, 54.97, 53.43, 52.57, 51.79, 48.72, 44.88, 34.93, 33.22, 32.51, 21.79. Synthesis of Hyperbranched Poly(MA-NMGA). A typical example (PAE-08 in Table 1) is given as follows. In a flask were placed 0.1 mol of NMGA and 30 mL of DMSO. When the mixture was completely dissolved, 0.105 mol of MA dissolved in 10 mL of methanol was dropped slowly
into the flask within 10 h, and the mixture was kept at room temperature for 50 h. Then the flask was connected to a revolving-distillation apparatus. Under reduced pressure, the residual MA and methanol were removed. Then 0.05 g of Zn(CH3CO2)2 was added to the flask. Under vigorous revolving and vacuum distillation, the mixture was kept at 60 °C for 1 h, 100 °C for 2 h, 120 °C for 2 h, and 145 °C for 2 h. The raw product was dissolved in 40 mL of DMSO and then poured into 1000 mL of acetone. The precipitate was collected and purified by reprecipitation from DMSO solution into acetone. A yellow plastic solid, 22.5 g (yield 79.9%), was obtained. IR (KBr): 3399.5 cm-1 (OH), 1731.5 cm-1 (CdO). 1H NMR (DMSO-d6, ppm): δ 4.6-4.2 (OH), 3.72 (NCH2CHO), 3.58 (CHO), 3.4 (CH2O), 2.68 (CH2N), 2.2 (CH3N), 1.6 (CH2CdO). 13C NMR (DMSO-d6, ppm): δ 175.4, 172.23, 84.46, 79.72, 79.02, 77.82, 77.4, 72.78569.92 (group), 68.85, 63.82, 62.12, 60.19, 58.98, 54.26, 53.81, 53.56, 52.4, 51.96, 51.22, 42.73, 42.52, 40.82, 37.14, 34.02, 31.84, 30.53, 29.9, 21.8. End-Capping Reaction. In a flask were placed 5 g of hyperbranched poly(MA-DEOA) and 30 mL of pyridine; then 4.2 g of benzoyl chloride was dropped slowly into the flask within 2 h, and the mixture was kept at 40 °C for 10 h. The mixture was poured into 500 mL of diethyl ether. The precipitate was collected and dried under vacuum. A powdery solid (7.1 g) was obtained. 1H NMR (DMSO-d6, ppm): δ 8.0-7.3 (-Ar-), 3.62 (ArCOOCH2), 3.45 (CH2O), 2.45 (CH2N), 1.6 (CH2CdO). General Procedure for Test of Degradation Property. To a flask was added 1 g of solid sample poly(MA-DEOA) in 100 mL of water. After the sample dissolved completely, the flask was placed in a water bath to keep the temperature of the sample at 37.5 °C. The pH of the solution was kept constant by adding acid or base. An aliquot of the solution was withdrawn every 10 h and analyzed by GPC. Results and Discussion Molecular Design of the Approach and Material. The classic approach to hyperbranched polymers is the polycondensation of ABn-type monomers, which has been studied theoretically by Flory19 as early as 1952 (Scheme 1). Being different from Flory’s approach, this work explored a new strategy to hyperbranched polymers from two monomers
706
Biomacromolecules, Vol. 4, No. 3, 2003
Scheme 1. General Approach to Hyperbranched Polymer
based on the principle of nonequal reactivity of different functional groups. Scheme 2 shows the design idea. Monomer AB contains one A and one B functional group, and they cannot react with each other. Monomer CDn has one C functional group and n D functional ones, and C cannot react with D. At room temperature, the B group of the AB monomer can easily react with the C group of CDn, but only at higher temperature and in the presence of catalyst, the A group can react with the D group. To obtain a water-soluble polymer that might be used as a drug delivery system, a secondary amino group (HN-) is selected as C group and hydroxyl (-OH) is selected as D group. Acrylate group (CH2dCHCO) plays the role of B group, which can react with secondary amino group under mild conditions. Methyloxy carbonyl (CH3OCdO) is chosen as A group because it can react with hydroxyl group at high temperature to form an ester unit. The ester unit is attractive in the synthesis of polymer materials because of its degradability. Here, ester Scheme 2. “AB + CDn” Approach to Hyperbranched Polymer
Gao et al.
units play another very important role to neutralize amines formed after degradation. Therefore, pH of the aqueous system can be self-adjusted to about 7, which is an important parameter for a good drug delivery system.23 In the reaction, Michael addition of C groups to B groups generates ADn-type species. Further polycondensation of the ADn species results in hyperbranched polyester with tertiary amino groups in backbone and end hydroxyl groups. So the novel approach is a “one-pot two-step” method. Analysis of Mass Spectrum at the First Stage. Scheme 3 shows the possible reactions between MA and DEOA. Compound 3 is the targeted molecule. Species 4 is not stable and will, if formed, further react with DEOA to form species 6. Molecule 6, having four hydroxyl groups, can play the role of “core molecule” in the preparation of hyperbranched polymers, which will limit the molecular weight.46,47 Species 5 is also not stable and will further react with DEOA and MA to form species 7, 8, and 9. Self-condensation of molecule 9, AD3-type monomer, would give hyperbranched polyester. So the occurrence of side reactions has no determined influence on the formation of hyperbranched polymer with high molecular weight. Figure 1 displays the mass spectrum of the mixture that was taken from the reaction system after removing the residual MA monomer. The peaks of m/z ) 192.3 and 214.2 are assigned to the ion peak of molecule 3 coupled with a proton (M + 1) and that of molecule 3 coupled with a Na+ (M + 23), respectively. The peaks of m/z ) 351.5, 510.5, and 691.7 are attributed to the molecular ion peaks of dimer (M2H+), trimer ((M3H+), and tetramer (M4H+) of 3, respectively. The peaks of m/z ) 373.5 and 532.5 are the
Biomacromolecules, Vol. 4, No. 3, 2003 707
Water-Soluble Degradable Hyperbranched Polyesters Scheme 3. Possible Reactions in the MA and DEOA System at Initial Stage
corresponding peaks of dimer and trimer of 3 with a captured Na+ (M2Na+, M3Na+). The fragment peaks of 3 are observed as two small peaks at m/z ) 118.2 and 174.3. Analysis of the mass spectrum shows that an AD2-type intermediate does form dominantly during the initial reaction stage. If monomer NMGA, instead of DEOA, is used to react with MA, the same result can be obtained from its corresponding mass spectrum. In Situ 1H NMR Spectra. The initial reaction process of monomer 1 and 2 was further monitored by in situ 1H NMR. Because monomer 2 contains amino and hydroxyl groups, diethylamine and 1,4-butanediol were used as models to react with monomer 1, respectively. Figure 2 displays the in situ 1 H NMR spectra of the reaction between methyl acrylate and diethylamine in solution of CD3OD. Michael addition of the secondary amino group of diethylamine to the double bond of methyl acrylate leads to the predicted product. The reaction between group A (CH3O-) and group C (-HN-) was not observed in the NMR spectra, which implied that species 4 and 6 showed in Scheme 3 were hardly generated at room temperature. On the other hand, the reaction between
Figure 1. Mass spectrum for MA and DEOA reaction system after removing residual MA. The reaction temperature is 25 °C.
group B (CH2dCHCO) and group C (-HN-) was fast within the initial several hours, and about 90% of monomers reacted with each other. However, a small quantity of two monomers still appeared in the 1H NMR spectra after 72 h, which indicated that the reaction approached its equilibrium. In situ 1H NMR spectra of the reaction between methyl acrylate and 1,4-butanediol are given in Figure 3. Almost no changes can be observed between the spectrum recorded at 5 min and the spectrum monitored at 48 h, which suggested that the reaction between group A and D was neglectable at room temperature. So the amount of species 5, 7, 8, and 9 described in Scheme 3 should be very small. Figure 4 shows the in situ 1H NMR spectra of the reaction between methyl acrylate (MA) and diethanolamine (DEOA). Similar to the reaction between methyl acrylate and diethylamine, the reaction between MA and DEOA is also fast within initial several hours, while tiny peaks attributed to original monomers are still present in the NMR spectrum after 240 h when the feed ratio of MA to DEOA was 1/1. This characterization and model reactions indicated that the reaction between group B and C has equilibrium, and the possible minor reactions given in Scheme 3 are neglectable at the tested temperature. Therefore, in our experiments the amount of MA is a little greater than that of DEOA so that the latter can be completely reacted. In fact, after small amount of MA was added to the same reaction system, the peaks assigned to DEOA disappeared with a relatively fast rate. Effect of Reaction Conditions on the Polymerization. Reaction conditions such as temperature, reaction time, and catalyst always have strong influence on the polycondensation. In our work, it is vitally important to find suitable reaction conditions to obtain soluble hyperbranched polymer with high molecular weight but no gel. The reaction
708
Biomacromolecules, Vol. 4, No. 3, 2003
Gao et al.
Figure 2. In situ 1H NMR spectra of the reaction system between methyl acrylate and diethylamine in CD3OD: (A) diethylamine; (B) 5 min; (C) 40 min; (D) 90 min; (E) 72 h.
conditions and results are given in Table 1. The molecular weight distribution of the resulting polymers is very narrow when compared with the theoretical prediction value. During the initial stage, higher reaction temperature would aggravate the side reactions B, C, and D, which would increase the contents of 4, 5, 6, and 7. Obviously, high content of 6 or 8 with multifunctional groups would endcap the vinyl groups of ADn to stop the further polycondensation of A with D, which would decrease the molecular weight of the resulting hyperbranched polymers. Figure 5 displays the mass spectrum of the first stage sample when the reaction temperature is 60 °C. Indeed, species 4 (m ) 159.2), 5 (m ) 159.2), 6 (m ) 264.3), 7 (m ) 245.3), and 8 (m ) 264.3) are present in the mass spectrum as the peaks at m/z ) 160.1, 265.1, 246.1, and 265.1. So the reaction temperature was set at room temperature for the first stage. During the second stage, temperature also has strong influence on the polymerization. At the last 2 h, temperatures of 120, 135, 150, or 165 °C were chosen as the reaction temperature. At the temperature below 135 °C, the hyperbranched polyester with high molecular weight cannot be obtained, and at the temperature above 165 °C, cross-linking was observed. Only at the region of 135-150 °C, high molecular weight polymer was synthesized successfully. Furthermore, the cyclic structure content in the hyperbranched polymer reported here is very low according to
such high molecular weight. Otherwise, molecular weight of the resulting polymers would be much lower.36 The effect of catalyst on the reaction is investigated in the paper. In the same reaction conditions, the molecular weight of the resulting polymers catalyzed by Ti(C4H9O)4 is larger than that catalyzed by Zn(OAc)2 (Table 1), which indicates that Ti(C4H9O)4 is a more efficient catalyst for the condensation reaction than Zn(OAc)2. Measurement of Molecular Weight. The hydroxylterminated hyperbranched polyesters are highly soluble in polar solvents such as water, methanol, N,N-dimethylformamide (DMF), and DMSO. The hydroxyl-terminated hyperbranched polymers were end-capped with benzoyl chloride to avoid aggregation in the measurements of molecular weight. In the 1H NMR spectrum of the end-capped product, the peak of hydroxyl groups at 4.08 ppm was not observed and the peaks assigned to benzene rings appeared at 7.38.0 ppm, which indicated that the hydroxyl groups were reacted with benzoyl chloride. The data in Table 1 showed that the influence of terminal hydroxyl groups on the measurement of molecular weight in water is not as great as prediction. The molecular weights measured with general GPC in water are in agreement with those measured with MALLS apparatus in THF if the molar mass of benzoyl was considered in the determination of the molecular weight of the end-capped polymers.
Water-Soluble Degradable Hyperbranched Polyesters
Biomacromolecules, Vol. 4, No. 3, 2003 709
Figure 3. In situ 1H NMR spectra of the reaction system between methyl acrylate and 1,4-butanediol in CD3OD: (A) 1,4-butanediol; (B) 5 min; (C) 48 h.
Loss-Weight Investigation. The polymerization process of MA and DEOA was monitored with the loss weight of the reaction system (Figure 6). When 0.1 mol of DEOA was used as one of the raw materials, 3.203 g of CH3OH would be lost theoretically as the reaction degree of CH3O groups reached 100%. In our experiment, 3.210 g of mass was lost, which is in good agreement with the theoretic value if experimental errors are considered. On the other hand, almost no mass lost was observed after 2 h at 150 °C for the reaction. So the reaction time during the last stage was set as 2 h in the experiments. Degree of Branching. Dendritic polymers have highly branched structures. The degree of branching (DB) is used to quantitatively describe their branched feature. A highly branched polymer has dendritic units (ND), linear units (NL), and terminal units (NT). DB is equal to the ratio of ND and NT to the total units (ND + NT + NL).35 The DB is 100% for dendrimers and
