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Quaternized Poly(propylene imine) Dendrimers as Novel pH-Sensitive Controlled-Release Systems Z. Sideratou, D. Tsiourvas, and C. M. Paleos* Institute of Physical Chemistry, N.C.S.R. “Demokritos”, 15310 Aghia Paraskevi, Attiki, Greece Received June 25, 1999. In Final Form: October 21, 1999 Quaternized poly(propylene imine) dendrimers have been prepared and investigated for their ability to function as pH-responsive controlled-release systems. These functionalized dendrimers are able to solubilize pyrene due to charge transfer complexation between the probe and the tertiary amino groups of the dendrimeric branches. The introduction of the quaternary ammonium groups at the external surface of the dendrimers affects the protonation titration profile as compared to the parent compounds leading to the release of the entrapped pyrene within a narrow pH region and basically when the intermediate nitrogens are protonated. These properties render these materials promising candidates for pH-responsive controlled-release systems possibly including prospected drug delivery applications.
Introduction
Scheme 1
1
Dendrimers are highly branched macromolecules with well-defined structures consisting of a central core, the intermediate repeating units, and the terminal functional groups. Repeating groups in the interior determine the microenvironment and consequently the solubilization properties of dendrimers while terminal groups primarily determine the solubility and chemical behavior of these compounds in solution. Following intensive research that has been conducted on the synthesis of dendrimeric compounds, the work is recently focusing on the functionalization of the external groups of basic dendrimers. A diversity of functionalized dendrimers2 has been prepared including those bearing carbohydrate moieties,3 chiral groups,4 mesogenic5 or nitroxide stable free radical moieties,6 and polymerizable7 and recognizable groups.8 The latter groups impart to dendrimers the property to organize at interfaces9 or form organized supramolecular structures.10 (1) Newkome, G. R.; Moorefield, C. N.; Vo¨gtle, F. Dendritic Molecules. Concepts, Syntheses, Perspectives; Wiley-VCH: Weinheim, 1996 and references therein. (b) Dvornic, P. R.; Tomalia, A. Macromol. Symp. 1994, 88, 123. (c) Ardoin, N.; Astruc, D. Bull. Soc. Chim. Fr. 1995, 132, 875. (d) Fischer, M.; Vo¨gtle, F. Angew. Chem., Int. Ed. Engl. 1999, 38, 884. (e) Bosman, A. W.; Janssen, H. M.; Meijer, E. W. Chem. Rev. 1999, 99, 1665. (2) Smith, D. K.; Diederich, F. Chem. Eur. J. 1998, 4, 1353. (b) Fre´chet, J. M. J. Science 1994, 263, 1710. (3) (a) Aoi, K.; Itoh, K.; Okada, M. Macromolecules 1995, 28, 5391. (b) Lindhorst, T. K.; Kieburg, C. Angew. Chem., Int. Ed. Engl. 1996, 35, 1953. (c) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; Jayaranam, N.; Stoddart, J. F. Angew. Chem., Int. Ed. Engl. 1997, 36, 732. (d) Ashton, P. R.; Boyd, S. E.; Brown, C. L.; Nepogodiev, S. A.; Meijer, E. W.; Peerlings, H. W. I.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 974. (e) Jayaranam, N.; Nepogodiev, S. A.; Stoddart, J. F. Chem. Eur. J. 1997, 3, 1193. (4) (a) Peerlings, H. W. I.; Meijer, E. W. Chem. Eur. J. 1997, 3, 1563. (b) Sanders-Hovens, M. T. H.; Jansen, J. F. G. A.; Vekemans, J A. J. M.; Meijer, E. W. ACS Polym. Mater. Sci. Eng. 1995, 73, 338. (c) Jansen, J. F. G. A.; Peerlings, H. W. I.; de Brabander-Van den Berg, E. M. M.; Meijer, E. W. Angew. Chem., Int. Ed. Engl. 1995, 34, 1206. (5) Ponomarenko, S. A.; Rebrov, E. A.; Bobrovsky, A. Y.; Boiko, N. I.; Muzafarov, A. M.; Shibaev, V. P. Liq. Cryst. 1996, 21, 1. (6) (a) Bosman, A. W.; Jansen, R. A. J.; Meijer, E. W. Macromolecules 1997, 30, 3606. (b) Cameron, J. H.; Facher, A.; Latermann, G.; Diele, S. Adv. Mater. 1997, 9, 398. (7) Moszner, N.; Vo¨lkel, T.; Rheinberger, V. Macromol. Chem. Phys. 1996, 197, 621. (8) (a) Newkome, G. R.; Woosley, B. D.; He, E.; Moorefield, C. N.; Gu¨ther, R.; Baker, G. R.; Escamilla, G. H.; Merrill, J.; Luftmann, H. Chem. Commun. 1996, 2727. (b) Vale´rio, C.; Fillaut, J.-L.; Ruiz, J.; Guittard, J.; Blais, J.-C.; Astruc, D. J. Am. Chem. Soc. 1997, 119, 2588.
Within the framework of dendrimers’ external groups functionalization and in order to show the applicability to develop novel drug delivery systems based on the concept of a dendritic box,11 the modification of the external primary amino groups of diaminobutane poly(propylene imine) dendrimers with 32 (DAB-32) or 64 (DAB-64) primary amino groups was undertaken by interacting these groups with glycidyltrimethylammonium chloride affording DAB-32Quat and DAB-64Quat, respectively. This is a facile, one-step reaction performed under mild experimental conditions in water (Scheme 1). Recent investigations have shown12 that DAB-32 and DAB-64, due to the basic and hydrophobic environment of their microcavities, incorporate hydrophobic guests including the well-known pyrene probe. Protonation of (9) Watanabe, S.; Regen, S. L. J. Am. Chem. Soc. 1994, 116, 8855. (10) (a) Stevelmans, S.; van Hest, J. C. M.; Jansen, J. F. G. A.; van Boxtel, D. A. F. J.; de Brabander-van den Berg, E. M. M.; Meijer, E. W. J. Am. Chem. Soc. 1996, 118, 7398. (b) Newkome, G. R.; Moorefield, C. N.; Baker, G. R.; Saunders: M. J.; Grossman, S. H. Angew. Chem., Int. Ed. Engl. 1991, 30, 1178. (c) Schenning, A. P. H. J.; Elissen-Roman, C.; Weener, J.-W.; Baars, M. W. P. L.; van der Gaast, S. J.; Meijer, E. W. J. Am. Chem. Soc. 1998, 120, 8199. (d) Zeng F.; Zimmerman, S. C. Chem. Rev. 1997, 97, 1681. (e) Kuzdzal, S. A.; Monnig, C. A.; Newkome G. R.; Moorefield, C. N. J. Chem. Soc., Chem. Commun. 1994, 2139. (f) Hawker, C. J.; Wooley, K. L.; Fre´chet, J. M. J. J. Chem. Soc., Perkin Trans 1 1993, 1287. (g) Baars, M. W. P. L.; Froehling, P. E.; Meijer, E. W. Chem. Commun. 1997, 1959. (11) Jansen, J. F. G. A.; de Brabander-van den Berg, E. M. M.; Meijer, E. W. Science 1994, 266, 1226. (12) Pistolis, G.; Malliaris, A.; Tsiourvas, D.; Paleos, C. M. Chem.s Eur. J. 1999, 5, 1440.
10.1021/la990829v CCC: $19.00 © 2000 American Chemical Society Published on Web 12/04/1999
Dendrimers as Controlled-Release Systems
internal nitrogens releases the encapsulated pyrene. In this manner a controlled-release system, responding to a rather broad pH region was obtained. It was expected that the introduction of the hydrophilic quaternary group not only would enhance water solubility of the resulting new dendrimeric compounds but also could modify their release properties. Apparently, the quaternary groups, due to their relative bulkiness coupled with extreme hydrophilicity, may act as barriers for the solubilized compounds, controlling their release from the interior of the dendrimer. Furthermore it should be mentioned that the quaternary groups possess bactericide, antifungal, and antimicrobial properties.13 Therefore the compounds under investigation provide a useful vehicle for drug delivery. Experimental Section Materials and Methods. Diaminobutane poly(propylene imine) dendrimers were purchased from DSM Fine Chemicals and used as received. Glycidyltrimethylammonium chloride was purchased from Fluka. Pyrene (Aldrich) was purified by recrystallization and sublimation. The amount of solubilized pyrene in dendrimeric aqueous solutions was determined by UV absorption spectroscopy on a Perkin-Elmer Lambda-16 spectrophotometer monitoring the peak of pyrene at 334 nm after subtracting the absorbance of the dendrimer solutions. Pyrene fluorescence spectra were recorded on a Perkin-Elmer LS-5B spectrophotometer, excitating at 335 nm. NMR spectra were obtained in CDCl3 (10-3 M) with a Bruker AC 250 spectrometer operating at 250 MHz for protons and 62.9 MHz for carbon-13. Synthesis of Quaternized Dendrimers. To 0.1 mmol of DAB-32 or DAB-64 dissolved in water, an aqueous solution of 3.3 or 6.5 mmol of glycidyltrimethylammonium chloride was added. The mixture was allowed to react for several hours at room temperature and subsequently subjected to dialysis with the appropriate membrane in order to remove the excess of glycidyltrimethylammonium chloride and possibly unreacted dendrimers. Thus, for the dendrimer originating from DAB-32 cut-off membranes of molecular weight 1200 and 5000 were employed, while for the dendrimer originating from DAB-64 1200 and 12 400 cut-off membranes were used. The dialyzed solutions were lyophilized and dried extensively over phosphorus pentoxide since the materials are very hygroscopic. The complete introduction of the quaternary moiety at the external surface of the parent dendrimers was confirmed by elemental analysis from the excellent carbon-to-nitrogen ratios. Thus, for C376H880N94O32Cl32 calcd C/N 3.43, found C/N 3.32; for C760H1776N190O64Cl64 calcd C/N 3.43, found 3.42. It should be noted that absolute values from elemental analyses were not satisfactory as might be expected for these very hygroscopic quaternaries. The completion of the reaction was also established by proton and carbon-13 NMR. Thus, R-CH2 and β-CH2 protons relative to the newly formed secondary amino group appear at 2.65 and 1.70 ppm, compared to 2.55 and 1.45 ppm, respectively, for the unfunctionalized dendrimers. Oxiran ring protons, a triplet at 3.22 ppm and a doublet at 2.95 ppm, are replaced by a quintet at 4.35 ppm (-CHOH-) and a doublet at 2.80 ppm (-NHCH2CHOH-) due to ring opening. The R-CH2 protons relative to the quaternary group appear as a triplet at 3.65 ppm and protons of the quaternary methyl groups at 3.25 ppm. Finally the protons of the primary amino groups at 1.25 ppm were absent in the spectra of quaternized dendrimers. The carbon-13 NMR peaks of R and β carbons relative to the primary amino group, at 40 and 31 ppm, are now replaced by peaks at 48 and 27 ppm, respectively. Oxiran ring carbons at 44 and 46 ppm are not observed, and new peaks appear at 54.5 and 66 ppm. The R-methylene and methyl groups attached at the quaternary center are observed at 71 and 56 ppm, respectively. Pyrene Solubilization in Dendrimers. Both compounds were dissolved in ethanol. Subsequently, ethanol was distilled off and the dry film obtained was dissolved in water while the amount of pyrene remaining insoluble was removed by centrifugation. (13) Martins, L. M. S.; Mamizuka, E. M.; Carmora-Ribeiro, A. M. Langmuir 1997, 13, 5583.
Langmuir, Vol. 16, No. 4, 2000 1767 Table 1. Solubility of Pyrene in Parent and Quaternized Derivativesa compound
[dendrimer], M
[Py], M
DAB-32 DAB-32Quat DAB-64 DAB-64Quat
2 × 10-3 2 × 10-3 1 × 10-3 1 × 10-3
2.0 × 10-5 1.1 × 10-5 2.1 × 10-5 1.3 × 10-5
a Molar concentrations of dendrimers represent weight equivalent quantities.
Titration Experiments. An aqueous pyrene solution was progressively added to an aqueous dendrimer solution (2.87 × 10-3 M DAB-32Quat or 2.8 × 10-3 M DAB-64Quat) of the same pyrene concentration in order to maintain the pyrene concentration constant throughout the titration. Pyrene fluorescence intensity and I1/I3 ratio were monitored. Additionally, for the titration experiments with hydrochloric acid, aqueous pyrene solutions containing 1 × 10-3 M DAB-32Quat or 5 × 10-4 M DAB-64Quat were employed. Pyrene concentration was lower than its solubility in water (8 × 10-7 M) in order to avoid crystal formation during the experiment.
Results and Discussion The reaction of glycidyltrimethylammonium chloride with the primary amino group of the dendrimers is performed at room temperature and resulted, as expected,14 in the introduction of R-hydroxyamine moiety together with the trimethylammonium group in these dendrimers. Thus, the hydrophilicity of the external surface of the dendrimers is enhanced and special care is needed in handling these materials. Solubilization and release properties of quaternized dendrimers were investigated by monitoring the fluorescence properties of pyrene, a well-known and extensively studied probe. The concentration of incorporated pyrene, as determined by absorption spectroscopy, for parent and quaternized dendrimers is shown in Table 1. The solubility of pyrene is increased by a factor of about 25 in the parent dendrimeric compounds and by about 15 for these quaternized derivatives. The observed increase of pyrene solubility results from its relatively effective encapsulation in dendrimers as conclusively established in our previous work.12 It is apparent that the size and the character of the end groups determine the extent of solubilization of pyrene inside dendrimers. Evidently the highly polar exterior disfavors the incorporation of higher concentrations of pyrene. In a titration-like addition of DAB-32Quat or DAB64Quat to an aqueous 2.85 × 10-7 M solution of pyrene, a significant quenching of its fluorescence intensity was observed and also a decrease of the I1/I3 ratio (Figure 1). The value of the latter parameter, which is dependent on the micropolarity of the medium,15 in this case, it reflects the environment of the microcavities. As shown for the parent dendrimeric compounds,12 the quenching of the fluorescence intensity was attributed to the proximity of pyrene with the tertiary amino groups. With the progress of titration, the I1/I3 ratio decreases until it reaches a value equal to ca. 0.90, which is close to that observed in a lipophilic medium. Additionally, a weak exciplex fluorescence appeared at approximately 480 nm, which is attributed to the formation of a charge-transfer complex between pyrene and the tertiary amino groups of dendrimer.16 Thus, pyrene is deeply incorporated inside the (14) March, J. Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 4th ed.; J. Wiley & Sons: New York, 1992; Chapter 10, p 416. (15) Malliaris, A. Int. Rev. Phys. Chem. 1988, 7, 95. (16) Lakowicz, J. R. Principles of Fluorescence Spectroscopy; Plenum Press: New York and London, 1983; p 8.
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Figure 1. Plot of F/F° (2, DAB-32Quat; 4, DAB-64Quat) and I1/I3 ratio (b, DAB-32Quat; O, DAB-64Quat) as a function of the concentration of dendrimer. F° is the total fluorescence intensity of a 2.85 × 10-7 M aqueous solution of pyrene and F is the measured fluorescence at various dendrimer concentrations.
microcavities in order to avoid contact with the hydrophilic quaternary groups. The presence of the repeating tertiary amino groups together with the secondary moieties imposes the investigation of pyrene release as a function of pH. The microcavities being covered with basic groups are susceptible to protonation which leads to an enhancement of the polarity of their microenvironment rendering therefore impossible the charge-transfer complexation of pyrene with the tertiary amino groups. For these two reasons the probe is prone to be released from the interior of the dendrimers. The fluorescence intensity as a function of pH obtained when hydrochloric acid was added to dendrimeric aqueous solutions, is shown in Figure 2. A gradual increase of pyrene fluorescence intensity was observed down to pH ) 4, while in the region between 4 and 2 a significant increase of fluorescence was seen, becoming approximately equal to that in pure water (Figure 2). In addition the variation of the I1/I3 ratio as a function of pH follows a similar trend (Figure 2). By the addition of the acid down to pH value of 4, the I1/I3 ratio slowly increases; at pH values between 4 and 2 this ratio increases substantially to a value of 1.5, which is very close to the value of pyrene in pure water (1.62). Pyrene fluorescence quenching is not observed since the protonated amino groups can no longer form charge-transfer complexes with pyrene. In this connection it has to be noted that in a blank experiment, addition of triethylamine to an aqueous pyrene solution quenches its fluorescence, whereas addition of HCl increases again the fluorescence intensity. In addition, the environment of the microcavities becomes more hydrophilic, leading to the release of pyrene in the aqueous medium, as previously established with unfunctionalized poly(propylene imine) dendrimers.12
Figure 2. Plot of I1/I3 ratio (a) and F/F° (b) of aqueous 3.87 × 10-7 M pyrene solutions containing DAB-64Quat (4) or DAB32Quat (2) as a function of pH.
The behavior of quaternized dendrimers is however differentiated from that of the starting compounds in that the release of pyrene is mainly observed within a narrow pH region compared to the broad pH range of the parent compounds. The titration curves of the quaternized compounds are shown in Figure 3. An inflection point is clearly observed at pH = 3.5 for DAB-32Quat and at pH = 4 for DAB-64Quat. This notable change in the release behavior may be rationalized by considering the dendrimer-acid titration profile, which is different from that of the parent dendrimers,17 as comparatively shown in Figure 3. It seems that the quaternary groups in a way seal the dendrimeric surface modifying the release properties of the dendrimers. Proton and carbon-13 NMR experiments have been employed to study the protonation sequence of the secondary and tertiary amino groups. Specifically, at pH values between 10 and 5, R-CH2 and β-CH2 relative to secondary amino group are gradually shifted from 2.65 to 3.25 ppm and from 1.70 to 2.15 ppm, respectively. Additionally, the proton of the -CHOH- moiety is shifted from 4.35 to 4.65 ppm, while the R-methylene protons of the core tertiary amino groups are shifted from 2.55 to 3.20 ppm. Between pH 7 and 5 the tertiary amino groups located in the intermediate region start protonating, their R-methylene groups shifting from 2.70 to 3.10 ppm. Their protonation is completed at pH = 2 where all R-CH2 and (17) Kabanov, V. A.; Zezin, A. B.; Rogacheva, V. B.; Gulyaeva, Zh. G.; Zansochova, M. F.; Joosten, J. G. H.; Brackman, J. C.; Vreekamp, R. H. ACS Polym. Mater. Sci. Eng. 1998, 79, 445.
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Langmuir, Vol. 16, No. 4, 2000 1769
protonating, the peaks of R-CH2 shifting from 54 to 52 ppm. When protonation was completed, at pH = 2, this peak is observed at 50.5 ppm. It is therefore concluded that protonation proceeds initially at the external secondary amino groups and at the core tertiary nitrogens, followed by the intermediate tertiary amino groups at lower pH values. These results are in agreement with the protonation mechanism of the poly(propylene imine) dendrimers reported recently.18 The significant release of the probe at pH 2-4 mainly follows the protonation of the intermediate tertiary amino moieties. Conclusions
Figure 3. Titration curves of the quaternized compounds (2, DAB-32Quat; 4, DAB-64Quat) and of the parent dendrimer (O, DAB-32).
β-CH2 relative to tertiary amino groups appear at 3.45 and 2.35 ppm, respectively. Analogous results were obtained by 13C NMR; the peaks of R-CH2 and β-CH2 relative to the secondary amino group are gradually shifted from 48 and 27 to 40 and 23 ppm, respectively, as the pH is lowered from 10 to 5. The peaks of the core R-CH2 relative to tertiary amino groups are shifted from 52 to 48 ppm. Additionally, at pH values between 7 and 5, the tertiary amino groups located in the intermediate region start
The introduction of the pharmacologically significant quaternary ammonium groups at the surface of the poly(propylene imine) dendrimers not only enhances the water solubility of pyrene, as also observed for the parent compounds, but also critically affects the protonation titration profile leading to the release of the entrapped molecule within a narrower pH region. This property renders these materials promising candidates for pHsensitive controlled-release systems and possibly provides a strategy for designing new drug delivery systems. Acknowledgment. This work was partially supported by the Brite Euram Project BRPR-CT94-0401. LA990829V (18) Koper, G. J. M.; van Genderen, M. H. P.; Elissen-Roma´n, C.; Baars, M. W. P. L.; Meijer, E. W.; Borkovec, M. J. Am. Chem. Soc. 1997, 119, 6512.
