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Surface Acetylation of Polyamidoamine (PAMAM) Dendrimers Decreases Cytotoxicity while Maintaining Membrane Permeability Rohit B. Kolhatkar, Kelly M. Kitchens, Peter W. Swaan, and Hamidreza Ghandehari* Center for Nanomedicine and Cellular Delivery, Department of Pharmaceutical Sciences, University of Maryland, Baltimore, Baltimore, Maryland. Received December 18, 2006; Revised Manuscript Received September 13, 2007
Improving the oral bioavailability of therapeutic compounds remains a challenging area of research. Polyamidoamine (PAMAM) dendrimers are promising candidates for oral drug delivery due to their well-defined compact structure, versatility of surface functionalities, low polydispersity, and ability to enhance transepithelial transport. However, potential cytotoxicity has hampered the development of PAMAM dendrimers for in vivo applications. In this article, we have systematically modified the surface groups of amine-terminated PAMAM dendrimers with acetyl groups. The effect of this modification on cytotoxicity, permeability, and cellular uptake was investigated on Caco-2 cell monolayers. Cytotoxicity was reduced by more than 10-fold as the number of surface acetyl groups increased while maintaining permeability across the cell monolayers. Furthermore, a decrease in nonspecific binding was evident for surface-modified dendrimers compared to their unmodified counterparts. These studies point to novel strategies for minimizing PAMAM dendrimer toxicity while maximizing their transepithelial permeability.
INTRODUCTION Polymeric drug delivery systems generally entail improved efficacy while concomitantly decreasing the dose limiting toxicity of therapeutically active compounds (1, 2). Due to ease of administration and patient compliance, oral delivery remains the preferred route of drug intake; however, the macromolecular nature of polymers oftentimes necessitates their administration by intravenous injection. Polyamidoamine (PAMAM) dendrimers, owing to their unique characteristics such as nanoscopic size range, high drug-loading capacity, low polydispersity, and predictable size and surface charge, have shown promise in drug delivery (1, 3–5). Depending on their surface properties and size, PAMAM dendrimers are transported across epithelial barriers to a higher extent than some of the conventional linear water-soluble polymers (6). Our recent data suggest that cationic PAMAM dendrimers may permeate across Caco-2 cells to a higher extent than their neutral and anionic counterparts (7). However, an important element limiting the clinical application of cationic dendrimers is their cytotoxicity, which is dependent on concentration, generation, and exposure time to the cellular barriers (8–12). Studies using epithelial cell lines have shown that the cytotoxicity of cationic dendrimers can be reduced by functionalizing their surface amine groups (13, 14). In this article, a small acetyl group was used for neutralization of surface amines to avoid the influence of molecular weight on the properties of the polymer. Other factors that prompted the use of acetyl groups for surface functionalization were as follows: (1) an increasing interest in surface-modified dendrimers for drug delivery; (2) ease of controlling the degree of acetylation by controlling stoichiometry; (3) mild reaction conditions required for acetylation; and (4) increased solubility of acetylated dendrimers. Two different generations of PAMAM dendrimers, namely, G2 and G4, were acetylated and the effect * Author for correspondence: Hamidreza Ghandehari, Ph.D., University of Maryland School of Pharmacy Center for Nanomedicine and Cellular Delivery, Department of Pharmaceutical Sciences, 20 Penn Street, HSFII Room 625, Baltimore, MD 21201-1075, USA. Telephone: (410) 706-8650. Fax: (410) 706-5017. E-mail address: hghandeh@ rx.umaryland.edu.
of surface modification on cytotoxicity, permeability, and uptake on Caco-2 epithelial cells was studied.
EXPERIMENTAL PROCEDURES Materials. PAMAM-G2NH2 (molecular weight ) 3214, 16 amine end groups), PAMAM-G4NH2 (molecular weight ) 14215, 64 amine end groups), Acetic anhydride, [14C]-mannitol (specific activity 50 mCi/mmol), D2O, and Triton X-100 were purchased from Sigma-Aldrich Co. (St. Louis, MO). 3H-Acetic anhydride was purchased from American Radiolabeled Chemicals Incorporation. Superose 12 HR 16/50 column was purchased from Amersham Pharmacia Biotech (Piscataway, NJ). Caco-2 cells were purchased from American Type Cell Culture (ATCC, Rockville, MD). WST-1 cell proliferation reagent was purchased from Roche Applied Science (Indianapolis, IN). Precast 4–20% gradient express gels for PAGE were obtained from ISC Bioexpress (Kaysville, UT). Tris-glycine-SDS (TGS) buffer (pH ) 8.3) was purchased from Invitrogen (Carlsbad, CA). 1H NMR and 13C NMR spectra were obtained using Varian 500 MHz FT NMR. The NMR solvent used was D2O unless otherwise indicated. All other chemicals used were analytical grade. Surface Acetylation of PAMAM Dendrimers. Conjugates are designated as GxAy where x represents the PAMAM dendrimer generation and y represents the number of acetamide groups on the surface (number of amine groups functionalized). PAMAM dendrimers were reacted with different molar ratios of acetic anhydride in the presence of excess triethylamine to quench acetic acid which was formed as a side product during the reaction (Figure 1) (15). A typical procedure for the synthesis of acetylated PAMAM dendrimers (G2A16) was as follows. G2-PAMAM dendrimer (0.100 g, 0.03 mmol) was dissolved in dry methanol (10 mL) followed by addition of acetic anhydride (0.062 g, 0.61 mmol) and triethylamine (0.12 g, 1.2 mmol). The solution was stirred for 12 h at room temperature, and methanol was then evaporated to obtain crude acetylated PAMAM dendrimers. The crude product was redissolved in water and purified by extensive dialysis against distilled water using dialysis membrane of 500 MWCO (Spectrum Laboratories, Inc., Rancho Dominguez, CA). Purified acetylated PAMAM
10.1021/bc0603889 CCC: $37.00 2007 American Chemical Society Published on Web 10/26/2007
Surface Acetylation of PAMAM Dendrimers
Figure 1. Synthetic scheme for acetylation of PAMAM dendrimers.
dendrimer was then freeze-dried, and solid product was stored in a refrigerator. G2A7, G4A32, and G4A60 were synthesized following a similar procedure with different molar equivalents of acetic anhydride. Characterization of Acetylated PAMAM Dendrimers. Acetylated PAMAM dendrimers were characterized by size exclusion chromatography (SEC) on a Superpose 12 column (Amersham Biosciences, Piscataway, NJ) using a fast protein liquid chromatography (FPLC) system with phosphate buffer (pH 7.4) as the mobile phase. Eluting molecules were detected using UV detector at 280 nm. The number of surface amine groups modified was determined from 1H NMR by calculating the ratios of integral values of the signal for upfield CH3 protons (1.8 ppm) and integral values of the signal for -CH2- protons from PAMAM dendrimer (2.2–4 ppm) (15). Figure 2A shows a typical NMR spectrum for acetylated PAMAM dendrimer. 1 H-NMR (D2O, 500 MHz) values for the surface-modified polymers were as follows: G2A16. δ ) 1.89 (48H, s, COCH3), 2.30–2.40 (56H, m, C-CH2-CONH), 2.53–2.58 (28H, m, N-CH2-CH2NH), 2.70–2.78 (56H, m, N-CH2-CH2-CO), 3.18–3.25 (88H, CONH-CH2). G2A7. δ ) 1.88 (21H, s, COCH3), 2.28–2.42 (56 H, m, C-CH2CONH), 2.50–2.60 (28H, m, N-CH2-CH2NH), 2.68–2.80 (56H, m, N-CH2-CH2-CO), 2.96–3.06 (18H, CH2NH2), 3.18–3.38 (70H, CONH-CH2). G4A60. δ ) 1.89 (180H, s, COCH3), 2.30–2.42 (248H, m, C-CH2-CONH), 2.56–2.68 (124H, m, N-CH2-CH2NH), 2.74–2.88 (248H, m, N-CH2-CH2-CO), 3.18–3.30 (376H, CONH-CH2). G4A32. δ ) 1.88 (96H, s, COCH3), 2.30–2.42 (248 H, m, C-CH2CONH), 2.50–2.60 (124H, m, N-CH2-CH2NH), 2.68–2.80 (248H, m, N-CH2-CH2-CO), 2.96–3.06 (64H, CH2NH2), 3.18–3.38 (312H, CONH-CH2). The dendrimers were further characterized using matrix assisted laser desorption–ionization-time of flight mass spectrometry (MALDI-TOF MS) on a Bruker Omniflex (Bruker Daltonics, Billerica, MA) to determine their molecular weights. A stock solution of the dendrimer was prepared by dissolving 1 mg of the sample in methanol. A 1 µL aliquot of the stock solution was mixed with 9 µL of matrix solution. The matrix solution used for characterizing G2 analogues consisted of saturated solution of R-hydroxycinnamic acid, and the matrix solution used for G4 analogues consisted of 10 mg/mL 2,5dihydroxybenzoic acid in 20:80 acetonitrile/ water containing 0.1% trifluoroacetic acid. The dendrimer matrix mixture was spotted on the target plate (1 µL per spot), and the solvent was allowed to evaporate under ambient conditions. Analysis was performed in the linear mode. The variations in charge ratio of the dendrimers were analyzed by native polyacrylamide gel electrophoresis (PAGE) and SDS PAGE on a BioRad vertical electrophoresis system (Power Pac 200, BioRad, CA, USA). For SDS PAGE analysis, TGS buffer (10×, pH ) 8.3) was diluted with water by a factor of 10 to prepare the running buffer. Each well was loaded with 2.0 µL sample solution comprising 1.0 µL of 1.0 mg/mL PAMAM dendrimer and 1.0 µL sample buffer Laemmli (Sigma, MO).
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Developed gels were stained with Bio-Safe Coomassie Blue and destained with water. Native PAGE analysis was performed following similar procedures but with reverse polarity, Tris– glycine buffer (pH ) 8.3) and methylene blue sucrose dye solution (50% sucrose, 1% methylene blue) were used as the loading buffer. The surface density of amine groups, SDa (i.e., the number of amine groups/nm2 of the surface), was calculated by dividing the number of amine groups on each dendrimer by its surface area. Surface area was calculated by assuming a spherical shape for the dendrimer and the RG (radius of gyration) values obtained from molecular modeling calculations for different-generation dendrimers (16). Synthesis and Characterization of Radiolabeled PAMAM Dendrimers.The surface modified G4 analogues (G4A32, G4A60) as well as unmodified G4NH2 PAMAM dendrimers were reacted with [3H]-labeled acetic anhydride (5 mCi/mmol of the polymer) as described above to obtain [3H]-labeled G4A32 and G4A60 PAMAM dendrimers. The crude product was purified by dialysis against distilled water for 48 h using dialysis membranes of 1000 MWCO (Spectrum Laboratories, Inc., Rancho Dominguez, CA). The polymers did not show any small molecular weight impurities as examined by size exclusion chromatography using a PD-10 column while measuring the radioactivity of the fractions collected. The specific activity for each radiolabeled polymer was in the range 2–3 mCi/mmol. Cell Culture. Caco-2 cells (passages 30–60) were grown at 37 °C in an atmosphere of 5% CO2 and 95% relative humidity. Cells were maintained in T-75 flasks using Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum, 1% nonessential amino acids, 10 000 units/mL penicillin, 10 000 µg/mL streptomycin, and 25 µg/mL amphotericin. Medium was changed every other day, and cells were passaged at 70–90% confluency using 0.25% trypsin/ethylenediamine tetraacetic acid (EDTA) solution. Incubation buffer consisted of Hank’s balanced salt solution (HBSS) supplemented with 10 mM N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) hemisodium salt (HEPES) buffer (pH 7.4). Cytotoxicity Assay. Caco-2 cells were seeded (50 000 cells/ well) in 96-well cell culture plates (Corning, Inc., Corning, NY) and maintained for 48 h. The cells were washed twice with HBSS and incubated with 100 µL of unmodified and modified PAMAM dendrimer solutions in HBSS. After 3 h, the cells were washed with HBSS to remove the dendrimers and replaced with 100 µL HBSS. Thereafter, 10 µL of cell proliferation reagent WST-1 was added to each well, followed by an incubation period of 4 h. Absorbance was measured at 440 nm using a SpectraMax 384 Plus plate reader (Molecular Devices, Sunnyvale, CA). HBSS was used as a negative control. The experiment was repeated 3 times in triplicate for each concentration of the PAMAM dendrimer. Cellular Uptake of PAMAM Dendrimers. To determine cellular uptake of radiolabeled PAMAM dendrimer, Caco-2 cells were seeded with a cell density of 80 000 cells per well on cell culture-treated 12-well plates (Becton Dickinson). After 48 h, cells were washed with HBSS and incubated with 0.01 mM radiolabeled PAMAM dendrimers for 30, 60, and 120 min, respectively. At the end of the incubation period, 20 µL of the sample of donor dendrimer solution was collected for measuring radioactivity, and the cells were washed twice with ice–cold HBSS buffer to stop the uptake process. The cells were then lysed with 1 N NaOH solution which was then neutralized with HCl. The cell-associated radioactivity was determined using liquid scintillation counting (Beckman Coulter, Fullerton, CA). The uptake data was normalized to total protein content determined using BCA protein assay kit (Pierce, Evanston, IL).
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Figure 2. (A) 1H NMR of G2A16. (B) MALDI TOF MS spectra for unmodified G2 (left) and G2A16 (right). (C) Native PAGE analysis of G2, G2A7, and G2A16 in lanes 1, 2, and 3, respectively. (D) Native PAGE analysis of G4A60, G4A32, and G4 in lanes 1, 2, and 3, respectively. (E) SDS PAGE analysis of G2, G2A7, G2A16, G4, G4A32, and G4A60 in lanes 1, 2, 3, 4, 5, and 6, respectively.
Transepithelial Transport Studies. Caco-2 cell monolayers were seeded (80 000 cells/well) on 12-well Transwell cell culture inserts (3 µM pore size). After the cells reach confluency (21–28 days in culture), the transport of PAMAM dendrimers from the
apical to the basolateral direction was investigated at donor concentrations of 0.01 mM for G4, 0.01 mM and 0.1 mM for G4A32, and 0.01 mM, 0.1 mM, and 1 mM for G4A60. Prior to experiments, the cells were washed with HBSS buffer, and then
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500 µL of the [3H]-labeled polymer was added to the apical side of each insert while 1500 µL of the HBSS buffer was added on the basolateral side. Samples were collected at 0, 30, 60, 90, and 120 min from the basolateral compartment and analyzed for accumulated radiolabel using a liquid scintillation counter (Beckman Coulter, Fullerton, CA). The activity of the apical donor solution was also measured at t ) 0 and 120 to assess mass balance and determine accurate donor concentrations. The apparent permeability (Papp) coefficients for PAMAM dendrimers were calculated as follows: dM Papp ) C0 · S · dt
(1)
where dM represents the change in dendrimer accumulation on the basolateral side, C0 the concentration on the apical side of the membrane, S the surface area of the insert (1.12 cm2), and dt the change in time. Monolayers were considered to be confluent when Papp for mannitol was less than 2 · 10 · 10-6 cm/s. Additionally, cell monolayer integrity was monitored during transport studies by using an epithelial voltohmmeter (EVOM) (World Precision Instruments, Inc., Sarasota, FL) to measure the transepithelial electrical resistance (TEER) at time points coinciding with sample collection. TEER values (Ω cm2) were corrected for background (obtained from inserts without cells) and normalized to untreated inserts (% control). After 120 min, the dosing solution was removed, and cells were washed with HBSS. Subsequently, cells were incubated in DMEM and placed at 37 °C overnight. After 24 h, TEER was measured to assess reversibility of tight junctional perturbance. In a different set of experiments using a similar procedure, the relative integrity of the paracellular (tight junctional) pathway was assessed by evaluating [14C]-mannitol (2.0 µM) permeability in the presence of PAMAM dendrimer. Statistical Analysis. Statistical analysis was carried out using Student’s t-test with a probability value p < 0.05 considered statistically significant.
RESULTS AND DISCUSSION Synthesis and Characterization of Acetylated PAMAM Dendrimers. This study is a logical extension of our previous investigation in which we demonstrated that the permeability of amine-terminated PAMAM dendrimers across Caco-2 cell monolayers is controlled by size, molecular weight, and concentration (4, 11). Subsequent studies indicated that cationic PAMAM dendrimers modulate tight junctions of Caco-2 cells, thereby enhancing the transepithelial transport of mannitol (7). However, the surface amine groups also render cationic PAMAM dendrimers cytotoxic, limiting their utility as safe and effective drug delivery systems (7, 12). With the hypothesis that surface modification can reduce cytotoxicity while maintaining optimal cellular uptake as well as transepithelial permeability, in this study 47% and 100% of the surface amine groups of G2 were functionalized to acetamide to yield G2A7 and G2A16, respectively. Likewise, 50% and 94% of the surface amine groups of G4 were functionalized to acetamide to yield G4A32 and G4A60, respectively. A typical 1H NMR spectrum obtained for completely modified G2A16 dendrimer is shown in Figure 2A. In the 1H NMR spectra, a downfield shift of the signal for CH2-NH2 and the appearance of a new signal for CH3 protons at 1.89 ppm, as well as the appearance of a new peak at 21.94 ppm corresponding to COCH3 in 13C NMR spectra (data not shown) indicate the formation of a covalent bond between surface amine and carbonyl carbon from acetic anhydride. MALDI-TOF MS analysis (Figure 2B) demonstrated an increase in the molecular weight of the polymer as the number of surface acetyl groups increased, suggesting the
Table 1. Characteristics of PAMAM Dendrimers Studied PAMAM dendrimer G2NH2 G2A7 G2A16 G4NH2 G4A32 G4A60
no. of surface elution surface NH2/acetyl Mw Mw volume density of groupsa calcd (MALDI) (FPLC) ml amine groupsb 16/0 9/7 0/16 64/0 32/32 4/60
3250 3550 3928 14215 15559 16735
3258 3563 3934 13337 14782 15887
14.85 14.77 14.74 13.10 12.94 12.76
0.60 0.34 0 0.71 0.35 0.04
a Determined from NMR data. b Calculated by dividing number of surface amine groups by surface area (nm2).
formation of a covalent bond rather than a complex. Similar MALDI-TOF spectra were obtained for all other unmodified and surface-modified PAMAM dendrimers. The estimated molecular weights (Table 1) were in good agreement with the NMR data obtained. Size exclusion analysis of the polymers showed the absence of small molecular weight impurities. A decrease in the elution volume of the polymers was observed as the number of surface acetyl groups increased (Table 1). Native PAGE analysis with reverse polarity showed that gel migration slowed as the positive charge on the polymer reduced (Figure 2C,D), whereas SDSPAGE analysis revealed that fully acetylated dendrimers migrated farther as compared to unmodified and partially acetylated dendrimers (Figure 2E). No differences were observed in bands of unmodified and partially acetylated dendrimers (Figure 2E). The bands in native page analysis were broad when compared to the narrow bands obtained from SDS-PAGE. The reason for this is that the native page separated compounds on the basis of charge and molecular weight whereas in SDS-PAGE separation is achieved on the basis of molecular weight only. Thus, in both native PAGE and SDS-PAGE analyses lower-generation smaller dendrimers (G2) migrated farther than higher-generation dendrimers (G4). Interestingly, in a subset of one generation subjected to native PAGE analysis, dendrimers with more positive charge showed farther migration as compared to dendrimers with a lower number of positively charged amine groups. As expected, fully acetylated dendrimers remained at the base due to lack of surface charge. Acetylation of PAMAM Dendrimers Reduces Cytotoxicity. An evaluation of the effect of dendrimer concentration and the relative degree of surface amine group acetylation on cell viability (Figure 3A) revealed a complete absence of cytotoxicity at the lowest concentration of all PAMAM dendrimers tested (0.01 mM). Increasing the concentration to 0.1 mM significantly decreased cell viability for unmodified dendrimers; however, cell incubation with either partially or fully acetylated PAMAM dendrimers resulted in greater than 90% cell viability. These data suggest that even partial surface modification at this concentration reduces cytotoxicity. Another 10-fold increase in dendrimer concentration (1 mM) demonstrated a concomitant drop in cell viability for unmodified dendrimers. Interestingly, cytotoxicity was absent in cells incubated with PAMAM dendrimers exhibiting the highest degree of acetylation, suggesting a linear relationship between the number of amine groups and cellular cytotoxicity. In fact, a simple plot of cell viability vs the number of surface amine groups (Figure 3B) suggests that cytotoxicity is dependent on the dendrimer generation, probably due to the intrinsic toxicity associated with higher generation dendrimers. However, when cell viability was plotted as a function of surface density (SDa), a linear correlation was observed irrespective of dendrimer generation (Figure 3C). Overall, G4 dendrimers showed lower cell viability (27%) as compared to G2 (56%) because of an increase in the surface density of amine groups, whereas G2A7 and G4A32 showed similar cell viability (73% vs 70%) due to
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Figure 3. (A) Viability of Caco-2 cells after incubation for 3 h with dendrimers at 1 mM, 0.1 mM, and 0.01 mM. Results are expressed as mean ( SEM (n ) 9). (B) Relationship between cell viability and number of surface amine groups for (9) G4 (R2 ) 0.99) and (2) G2 analogues (R2 ) 1). (C) Relationship between cell viability and surface density (R2 ) 0.93).
similar surface density (0.34 vs 0.35) of amine groups. Previously, various investigators (17–19) found that cytotoxicity of water-soluble cationic polymers is governed by molecular weight, charge density, and the type of amine functionality (primary vs tertiary). Ryser (18) suggested a three-point attachment for eliciting a biological response on the cell membrane and speculated that the cytotoxicity of a polymer will decrease when the space between reactive amine groups is increased. In the current report, we have tried to quantify these observations with PAMAM dendrimers. A multiregressional analysis (F < 0.001; R2 ) 0.7) using cell viability, %CV, as a dependent variable with surface density, SDa, and concentration, C, as independent variables revealed the following relationship: %CV ) -15.5 logC - 50.9SDa + 87.6
(2)
Equation 2 suggests that cell viability in the presence of amine-terminated PAMAM dendrimers is influenced by concentration, size, and number of surface amine groups. This mathematical expression can be useful in predicting the appropriate surface modification required to minimize toxicity of PAMAM dendrimers in the size range studied. Acetylated PAMAM Dendrimers Retain Cellular Permeability Comparable to Unmodified Dendrimers. The permeability of native and surface-modified G4 analogues was evaluated across Caco-2 cell monolayers (Figure 4A). At previously determined nontoxic concentrations (0.01 and 0.1 mM; Figure 3A), we observed a significant increase in [14C]mannitol permeability (Figure 4B) and a reduction in TEER values (Figure 4C). Since mannitol is a compound that exclusively traverses the cell monolayer via the paracellular pathway, these data indicate that PAMAM dendrimer incubation led to modulation of the epithelial tight junctions. As further evidence that incubation with acetylated PAMAM dendrimers
Figure 4. (A) Permeability of G4NH2, G4A32, and G4A60 across Caco-2 cell monolayers after 120 min. Results are reported as mean ( SEM (n ) 6). (*) (p < 0.05) and (**) (p < 0.01) denote a significant difference in permeability when compared to permeability of unmodified PAMAM dendrimer at 0.01 mM. (×) Permeability is not evaluated due to cytotoxicity. (B) Permeability of [14C]-mannitol across Caco-2 cell monolayer in the absence and presence of G4NH2, G4A32, and G4A60 dendrimers. Results expressed as mean ( SEM (n ) 6). (×) Permeability is not evaluated due to cytotoxicity. (C) Effect of unmodified and surface-modified PAMAM dendrimers on transepithelial electrical resistance (TEER): (9) control, (1), G4NH2 (0.01 mM), (2) G4A32 (0.01 mM), (O) G4A32 (0.1 mM), ()) G4A60 (0.01 mM), (0) G4A60 (0.1 mM), (•) G4A60 (1 mM). Results expressed as mean ( SEM (n ) 6). For clarity, error bars are not shown in the figure.
does not lead to permanent cell damage, we observed the full reversal of tight junctional modulation, as demonstrated by cellular TEER values, after a 24 h equilibration period (Figure 4C). Permeability enhancement was a function of both dendrimer surface acetylation and donor concentration. At 0.01 mM, a significant increase in the permeability (p < 0.05) of G4A32 was observed when compared to unmodified G4 dendrimer. However, an additional increase in acetylation levels (e.g., G4A60) did not further enhance permeability. Although unmodified G4 is toxic at 0.1 mM, 1.5- and 2-fold enhancements of cellular permeability can be achieved by increasing the G4A32 and G4A60 concentration from 0.01 to 0.1 mM, respectively (Figure 4B). Paradoxically, permeability at 1 mM G4A60 led to a significant decrease in its permeability, which
Surface Acetylation of PAMAM Dendrimers
Figure 5. Cellular uptake (pmol of dendrimer/mg of protein) of 0.01 mM G4, G4A32, and G4A60 at 30, 60, and 120 min.
cannot be ascribed to intrinsic cytotoxicity and needs to be further addressed. Overall, the highest level of permeation enhancement was achieved at 0.1 mM with partially acetylated G4. It is interesting to note that the permeation enhancement of acetylated dendrimer is strongly correlated with [14C]mannitol permeability, but not to concomitant linear changes in TEER values. This would suggest that surface-modified PAMAM dendrimers do not enhance their permeability via the tight junctional route alone, but their enhanced cellular translocation may entail a combined effect on paracellular as well as transcellular permeation pathways. Previous observations have shown that positively charged dendrimers exert higher permeability when compared to neutral and negatively charged dendrimers. Contrary to expectations, however, surface-modified dendrimers retained their permeability relative to unmodified dendrimers. Whereas this observation is unexpected, it should be noted that the effect of charge on permeability of dendrimer generation is generally more pronounced for lower-generation dendrimers (G2) (7). Alternatively, sustained permeability of surface-modified PAMAM dendrimers could be the result of a decrease in hydrophilicity of the polymers and/or conformational changes. Modification of the amine groups to amides renders the dendrimer surface neutral, thereby reducing the repulsive forces between amine groups and potentially leading to a more compact structural assembly. Another possible explanation for the increase in the permeability of G4A32 could be a reduction in nonspecific binding. Quintana and co-workers (20) reported that acetylation can be used to reduce nonspecific binding associated with amineterminated nanoparticles. A decrease in the number of positively charged surface groups may reduce the nonspecific interaction of acetylated PAMAM dendrimers with the negatively charged cell membrane. This, in turn, may result in a relatively higher concentration of free dendrimer at the apical side of the membrane contributing to increased permeability through the paracellular route. These data are in good agreement with studies by Wiwattanapattapee and colleagues (6), who observed higher tissue uptake and lower serosal transfer rate for cationic dendrimers compared to anionic dendrimers. In addition, highergeneration dendrimers (G4) used in the current study will still carry a large number of internal tertiary amine groups, which can potentially interact with negatively charged cell membranes, enhancing permeability. To further investigate their cellular entry mechanism, the uptake into Caco-2 cells of G4 surface-modified dendrimers was determined at nontoxic concentrations (0.01 mM). Assuming steady-state conditions after 30 min of equilibration, we found linear cellular accumulation of dendrimer with increasing incubation time (Figure 5), with a Papp (cm/s) of 1.7 · × 10-6,
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3.0 · × 10-6, and 2.1 · × 10-6 for G4NH2, G4A32, and G4A60, respectively. Although a significant decrease (p < 0.05) in initial uptake for acetylated G4 dendrimer was observed when compared to native dendrimer, there was no apparent decrease in permeability for G4A32 and G4A60, respectively. These data indicate that surface modification of cationic PAMAM dendrimers leads to only modest decreases in cellular uptake compared to native dendrimer, but results in a significant decrease in dendrimer-associated cytotoxicity. Therapeutic agents are often conjugated to PAMAM dendrimers to gain an overall improvement of their bioavailability. Such surface modification can change cellular permeability characteristics and contribute to changes in the transepithelial transport of PAMAM dendrimers across intestinal cell lines. The remaining surface amine groups available on partially acetylated dendrimers can be used for drug loading and conjugation of targeting moieties to the PAMAM dendrimers while effectively reducing their cytotoxicity in parallel. Thus, the observations from the current study indicate that surface modification of PAMAM dendrimers is a strategy that can be utilized for oral drug delivery.
CONCLUSIONS In this study, we differentially surface modified a series of cationic amine-terminated PAMAM dendrimers with acetyl groups. Although cationic dendrimers display concentrationdependent cytotoxicity, we observed a significant reduction of cellular toxicity with an increase in surface modification. Cell viability was strongly correlated with surface density of amine groups regardless of dendrimer generation. Surface-modified PAMAM dendrimers displayed permeability and uptake profiles similar to those of unmodified (native) dendrimer. Thus, acetylated PAMAM dendrimers feature reduced cytotoxicity while maintaining appreciable permeability. Taken together, these results suggest that it is possible to tailor-make surfacemodified PAMAM dendrimers for specific oral drug delivery needs.
ACKNOWLEDGMENT Authors appreciate the efforts of Dr. Julie A. Ray for assisting with MALDI-TOF MS experiments. Financial support for Rohit Kolhatkar was made possible by Department of Defense (Multidisciplinary Postdoctoral Fellowship W81XWH-06-10698) and for Kelly Kitchens from National Institute of General Medical Sciences (Predoctoral National Research Service Award F31-GM67278). Support was also provided by NIH R01 EB007470.
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