http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2014.908899
RESEARCH ARTICLE
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Preparation and in vitro characterization of pluronic-attached polyamidoamine dendrimers for drug delivery Zhuojun Gu1*, Meng Wang2*, Qiongyan Fang1, Huaiyu Zheng1, Feiyue Wu1, Dai Lin1, Ying Xu1, and Yi Jin2 1
Zhoushan Hospital, Zhoushan, P.R. China and 2College of Pharmaceutical Sciences, Institute of Pharmaceutics, Zhejiang University, Hangzhou, P.R. China Abstract
Keywords
Context: Polyamidoamine (PAMAM) dendrimers have attracted lots of interest as drug carriers. And little study about whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems has been carried on. Objective: Pluronic F127 (PF127) attached PAMAM dendrimers were designed as novel drug carriers. Methods: Two conjugation ratios of PF127-attached PAMAM dendrimers were synthesized. 1H nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectrum (FTIR), element analysis and ninhydrin assay were used to characterize the conjugates. Size, zeta potential and critical micelle concentrations (CMC) were also detected. And DOX was incorporated into the hydrophobic interior of the conjugates. Studies on their drug loading and drug release were carried on. Furthermore, hemolysis and cytotoxicity assay were used to evaluate the toxicity of the conjugates. Results and discussion: PF127 was successfully conjugated to the fifth generation PAMAM dendrimer at two molar ratios of 19% and 57% (PF127 to surface amine per PAMAM dendrimer molecular). The conjugates showed an increased size and a reduced zeta potential. And higher CMC values were obtained than pure PF127. Compared with unconjugated PAMAM dendrimer, PF127 conjugation significantly reduced the hemolytic toxicity and cytotoxicity of PAMAM dendrimer in vitro. The encapsulation results showed that the ability to encapsulate DOX by the conjugate of 19% conjugation ratio was better than that of 57% conjugation ratio. And the maximum is 12.87 DOX molecules per conjugate molecule. Moreover, the complexes showed a sustained release behavior compared to pure DOX. Conclusion: Findings from the in vitro study show that the PF127-attached PAMAM dendrimers may be potential carriers for drug delivery.
Conjugate, DOX, drug delivery, PAMAM dendrimer, pluronic F127, toxicity
Introduction Dendrimers are unique synthetic macromolecules with highly branched structure and globular shape1. These have definite molecular weight, shape, size and host–guest entrapment properties, which make them be ideal carriers for drugs and genes. Poly(amidoamine) (PAMAM) dendrimers have been widely studied and used among the dendrimers so far. The PAMAM dendrimers could be modified with multiple functionalities such as drugs, genes, targeting molecules and imaging agents for the plentiful surface groups2–6. In our previous study, PAMAM– triamcinolone acetonide (PAMAM–TA) conjugates were synthesized for efficient translocation of pDNA into the nucleus7.
*These two authors contributed equally to this work. Address for correspondence: Yi Jin, College of Pharmaceutical Sciences, Institute of Pharmaceutics, Zhejiang University, Hangzhou 310058, P.R. China. Tel/Fax: +86 571 88208435. E-mail: [email protected] Zhuojun Gu, Zhoushan Hospital, Zhoushan 316000, P.R. China. Tel/ Fax: +86 580 2292847. E-mail: [email protected]
History Received 19 June 2013 Revised 24 October 2013 Accepted 9 March 2014 Published online 18 April 2014
And the conjugates showed high transfection efficiency and low toxicity. Many PEGylated dendrimeric systems have been explored as potential drug delivery agents at present8,9. The PEG conjugation can increase drug loading and overcome a few drawbacks like hemolytic toxicity, drug leakage and so on10. In the previous studies, PEGylated PAMAM dendrimers can load drugs by two ways. One way is by conjugation with drugs through covallent bond11,12. And another way is by physical encapsulation with drugs13,14. For example, Zhu et al.15 prepared RGD-modified PEGPAMAM-DOX conjugate for targeted cancer therapies. And, Kojima et al.16 prepared M-PEG-attached PAMAM dendrimers to encapsulate DOX by the hydrophobic interior or MTX by the basic interior. While little study about whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems has been carried on. So the conjugation of PAMAM by pluronic for drug delivery would be an attractive field to research. Pluronic is a kind of polymer surfactants, which has a triblock structure: PEO-PPO-PEO. Pluronic is amphiphilic for the hydrophilic poly(ethylene oxide) (PEO) blocks and the hydrophobic poly(propylene oxide) (PPO) blocks. Pluronic has the ability to
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interact with biological membranes and to enhance transport of compounds into the cells17. And, it has been widely used in micelles, nanoparticles, hydrogels and so on18–21. Pluronic F127 (PF127) was used for its biocompatibility and its approval by FDA22. And DOX-loaded L61/F127 micelles were evaluated in Phase II study in 200223. In our present work, two conjugation ratios of PF127-attached PAMAM dendrimers were synthesized. Furthermore, DOX was chosen as a hydrophobic model drug and incorporated into the hydrophobic interior of the conjugates. Studies on their toxicity, drug loading and drug release were carried on to evaluate whether the conjugates could be potential drug carriers. The new drug carriers were expected to incorporate much more DOX and show better release behavior with decreased toxicity.
Materials and methods Materials Pluronic F127 was kindly supplied by BASF China Ltd. (Shanghai, China). The fifth generation PAMAM dendrimer was purchased from Weihai City Chenyuan Silicone New Material Co., Ltd. (Weihai, China). 4-Nitrophenyl chloroformate and 3-(4, 5-dimethylthi-zaol-2-yl)-2, 5-diphenyltetrazolum bromide (MTT) were purchased from Sigma–Aldrich China Inc. (Shanghai, China). Doxorubicin hydrochloride (DOXHCl) was purchased from Haikou Manfanyuan Chemical Co., Ltd. (Haikou, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biologic Co., Ltd. (Hangzhou, China). RPMI1640 medium (RPMI), trypsin-EDTA solution (0.5% trypsin, 5.3 Mm EDTA tetrasodium) and PBS (phosphate-buffered saline) were purchased from Jinuo Biomedical Technology Co., Ltd (Hangzhou, China). Distilled water used in the synthesis was prepared by Milli-Q (Millipore, Billerica, MA). All other chemicals were analytical grade. Activation of pluronic F127 Pluronic F127 (PF127) was activated with 4-nitrophenyl chloroformate as described earlier24. 4-Nitrophenyl chloroformate (0.4 mmol) was dissolved in benzene and then added to PF127 (0.4 mmol) dissolved in benzene drop by drop. Then the reaction mixture was stirred for 24 h at room temperature under a nitrogen atmosphere. After reaction, the activated PF127 was precipitated three times using 8-fold excess of petroleum ether (bp 30–60 C) to remove the unreacted 4-nitrophenyl chloroformate. By centrifugation, the activated PF127 was recovered and kept under vacuum overnight to remove the remaining solvent. The product was analyzed with 1H nuclear magnetic resonance (NMR) (Bruker AVIII500M, Fa¨llanden, Switzerland) and Fourier transform infrared spectrum (FTIR) (JASCO FT/IR4100, Tokyo, Japan). D2O was purchased from Aladdin (Shanghai, China) as a NMR solvent. Synthesis of PF127-attached PAMAM dendrimers About 1.3 mmol of the fifth generation PAMAM dendrimer was dissolved in dimethyl sulfoxide. The activated PF127 was added into the solution at different molar ratios (25% and 65% per PAMAM, respectively). Then the solution was stirred to react for 4 or 6 days at room temperature (depending on the PF127 modification ratio). After reaction, the solution was dialyzed against distilled water for 3 or more days. Finally, the PF127attached PAMAM dendrimers were obtained by lyophilization. The products were analyzed with 1H-NMR and FTIR. Element analysis (ThermoFinnigan Flash EA 1112, Milan, Italy) was measured to determine the exact number of the pluronic chains conjugated to the PAMAM dendrimer. The average size and zeta
Drug Dev Ind Pharm, Early Online: 1–7
potential values were measured with a Zetasizer (Malvern NANO ZS90, Worcestershire, UK). Ninhydrin assay Ninhydrin assay was used to determine the primary amine contents of the conjugates. The ninhydrin reagent was prepared on the day of the assay and used in 24 h. About 0.4 g ninhydrin and 0.06 g hydrindantin were dissolved in 15 ml of dimethyl sulfoxide followed by adding 5 ml of sodium acetate buffer (4 M) while flushing with nitrogen. The reagent was stored in 4 C. About 0.5 ml of the reagent was added to 0.5 ml of the sample. The tubes were capped immediately, then shaken briefly by hand and heated in a boiling water bath for 30 min. When the tubes were cooled down, 9 ml of 50% alcohol was added to each tube and the solutions were mixed with a vortex mixer for 15 s. The absorbance of each solution was measured with a UV-Vis spectrophotometer (Persee TU-1800PC, Beijing, China) at 570 nm. CMC of the PF127-attached PAMAM dendrimers Critical micelle concentrations (CMC) of the conjugates were determined to prove the potential of micelle formation. Fluorescence measurements were carried out by using pyrene as a probe to determine the CMC. Pyrene in acetone was added to a series of volumetric flasks, and then the solvent was evaporated. Different volumes of the conjugates were added to the volumetric flasks and diluted with distilled water separately. The final concentration of pyrene was 6.0 107 M, and the concentrations of the conjugates varied from 1.0 106 to 1.0 mg/ml. The solutions were incubated at 37 C with mild shake overnight to equilibrate the pyrene and the conjugates, and finally the solutions were incubated at room temperature. Fluorescence spectra were measured by a fluorescence spectrophotometer (JASCO FP 6500, Tokyo, Japan). Excitation spectra were obtained from the scanning excitation spectra from 300 to 360 nm, fixing the emission wavelength at 390 nm. Encapsulation of DOX by PF127-attached PAMAM dendrimers About 3.2 mg DOXHCl was dissolved in 1.5 ml methanol: acetone (1:1), and 5 ml triethylamine was further added to transform DOXHCl into hydrophobic DOX (2 mg/ml). PF127attached PAMAM dendrimers of equivalent molar were dissolved in PBS (pH 7.4), and then mixed with the same volume of the above DOX solution separately. The DOX was excess compared to the conjugates. The mixed solution was stirred for 24 h to evaporate the organic solvent. Then the solution was centrifuged at 13 000 g for 10 min to remove the unloaded DOX. Appropriate amount of the supernatant was diluted with PBS (pH 7.4) and absorbance was measured to determine the content of DOX with a UV-Vis spectrophotometer at 479 nm. Finally the supernatant was lyophilized to get the PF127-attached PAMAM dendrimer–DOX complexes. Release of DOX from PF127-attached PAMAM dendrimer–DOX complexes PF127-attached PAMAM dendrimer–DOX complexes were dispersed in 2 ml PBS (pH 7.4) separately before transferred into the dialysis bag (MWCO ¼ 4000). Then each bag was placed in 20 ml PBS (pH 7.4), and shaken at a rate of 100 g at 37 C. At the predetermined time intervals, 2 ml of the release medium was taken out, and 2 ml of fresh medium was added after each collection. The absorption of the release medium was measured by a UV-Vis spectrophotometer at 479 nm.
DOI: 10.3109/03639045.2014.908899
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In vitro hemolysis assay The hemolytic activity of the PAMAM and conjugated PAMAM dendrimers was investigated. Blood of New Zealand white rabbit was collected in heparinized tubes and centrifuged at 3000 g for 10 min. The cell pellet was washed three times with cold PBS (pH 7.4) by centrifugation at 3000 g for 10 min each time and finally resuspended in PBS (pH 7.4) to reach a concentration of 1.5% (v/v). The suspension of red blood cells was freshly prepared and used within 24 h after collection. Solutions of PAMAM dendrimer and PF127-attached PAMAM dendrimers were prepared in PBS (pH 7.4) and diluted to different concentrations. The polymer solutions were mixed with equal volume of the erythrocytes separately. The mixtures were incubated at 37 C for 4 h and centrifuged at 13 000 g for 10 min. Absorption of the supernatant was measured by a UV-Vis spectrophotometer at 540 nm to determine the release of hemoglobin. About 0.2% Triton X-100 was regarded as the positive control and PBS (pH 7.4) was regarded as the negative control.
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In vitro cytotoxicity assay The cytotoxicity of the PAMAM and PF127-attached PAMAM dendrimers was evaluated by MTT assay. MCF-7 cells were seeded into a 96-well plate at a density of 5 103 cells per well in 100 ml of culture medium and allowed to attach for 24 h. Then the cells were incubated with 25 ml of various determined concentrations of conjugates. After 48-h incubation, 31.5 ml of MTT solution (5 mg/ml in PBS) was added to every well and the cells were incubated for another 4 h. Then the culture medium was completely removed and 200 ml DMSO was added. Absorbance of each well was measured with a Microplate Reader (Bio-Rad, Hercules, CA) at 570 nm.
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Statistical analysis Data which were expressed as mean ± SD were repeated at least three times. Statistical significance was determined using Student’s t-test.
Results and discussion Synthesis and characterization of activated PF127 and PF127-attached PAMAM dendrimers PF127 was activated with equimolar amount of 4-nitrophenyl chloroformate to synthesis PF127 with the single hydroxyl end activated. So the number of both hydroxyl ends of PF127 activated was minimized. The characterization of the activated PF127 and PF127-attached PAMAM dendrimers was determined by 1H-NMR in D2O (Figure 1). The degree of activation was 81.35% from the peak intensity ratio of the aryl protons of the nitrophenyl groups (NO2–C6H4–, d ¼ 6.9–8.4 ppm) to the methyl protons of PF127 (–CH3, d ¼ 1.19 ppm) (Figure 1B). The peak in PF127-attached PAMAM dendrimers at 1.19 ppm corresponds to the methyl group (–CH3) of the PF127 (Figure 1A). And peaks belonging to PAMAM (Figure 1C) at 2.46 (–NCH2CH2CO–), 2.63 (–CONHCH2CH2N–), 2.76 (–CONHCH2CH2N), 2.83 (–NCH2CH2CO–), 3.27 and 3.37 (–CONHCH2CH2NH2, NCH2CH2NHCO–), are still observed in the conjugates (Figure 1D and E), which confirmed the formation of conjugation. A little shift of the peaks was shown in the spectra because of the conjugation of PF127 to the PAMAM dendrimer. No peaks of the aryl protons of the nitrophenyl groups (NO2–C6H4–, d ¼ 6.9-8.4 ppm) were observed in the spectra of the conjugates. So pure conjugates were got without activated PF127 (APF127) left.
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Figure 1. 1H nuclear magnetic resonance (NMR) spectra of PF127 (A), APF127 (B), PAMAM (C), PAMAM-n1 PF127 (D) and PAMAM-n2 PF127 (E).
FTIR was also used to characterize the activated PF127 and PF127-attached PAMAM dendrimers. FTIR spectra were recorded in the range between 4000 and 400 cm1 as shown in Figure 2. New absorption at 1770 cm1 belonging to the stretching vibration frequency of C ¼ O of eater group was shown in the spectrum of activated PF127. So the PF127 was successfully activated. Compared the FTIR spectra of the conjugates with that of PF127, new absorptions at 1649
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Figure 2. FTIR spectra of PF127, APF127, PAMAM-n1 PF127 and PAMAM-n2 PF127. Table 1. PF127 conjugation ratios of PF127-attached PAMAM dendrimers determined by element analysis.
Products PAMAM-n1 PF 127 PAMAM-n2 PF127
Theoretical PF127 conjugation ratios (%)
Detected carbon (%)
Detected nitrogen (%)
The theoretical number of PF127 chains
The detected number of PF127 chains
Detected PF127 conjugation ratios (%)
25% 65%
55.53 56.65
2.09 0.75
32.00 83.20
24.28 72.94
18.97 56.98
and 1555 cm1 were shown in the spectra of the conjugates, and absorption at 1770 cm1 was not observed. Absorption at 1649 cm1 belongs to the stretching vibration frequency of C ¼ O of secondary amide group, and absorption at 1555 cm1 belongs to the stretching vibration frequency of C–N and the bending vibration of NH of secondary amide group of the products. So the same conclusion could be drawn that the conjugates were successfully synthesized without APF127 left. Element analysis was used to analyse the ratio of C, N and H elements of the final products and the results were listed in Table 1. The exact number of the PF127 chains conjugated to the PAMAM dendrimer was got through the C/N ratio. The added APF127 was not fully conjugated to the PAMAM dendrimer. Even after a long reaction time, PAMAM-25% PF127 achieved a PF127 conjugation ratio of 19%, and PAMAM-65% PF127 achieved a PF127 conjugation ratio of 57%. One of the reasons is that a further reaction between the APF127 and the primary amine groups of PAMAM dendrimer could be blocked because of the long and convoluted chain of PF127. And similar results were achieved in the process of PEG conjugation to PAMAM dendrimers3,25. PAMAM-n1 PF127 and PAMAM-n2 PF127 were used to represent the final products (n represents the conjugation ratio, and n15n2). Determination of primary amines The absorption intensity of the solution is proportional to the amount of free amines. The primary amine contents of the dendrimers were determined by ninhydrin assay. As Figure 3 shown, much lower absorption intensity of the PF127-attached PAMAM dendrimers than PAMAM dendrimer was observed at the same concentration. The primary amine density of PAMAM decreased for the conjugation of PF127 to the PAMAM dendrimer. As for the different conjugation ratios of the conjugates, the absorption decreased as the conjugation ratios increased. The absorption intensity of PAMAM-n2 PF127 was lower than that of PAMAM-n1 PF127, so a conclusion could be drawn that the value of n2 is bigger than n1. Thus the different
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Figure 3. UV curves of PAMAM, PAMAM-n1 PF127 and PAMAM-n2 PF127 for ninhydrin assay.
absorption intensity of the conjugates could reflect the degree of conjugation. CMC of the PF127-attached PAMAM dendrimers PF127 is amphiphilic for its PEO-PPO-PEO structure. It has the ability to form micelles in aqueous solutions above the critical micelle concentration. To demonstrate that the PF127-attached PAMAM dendrimers could also form micelles above the CMC, a fluorescent measurement was taken by using pyrene as a probe. Pyrene has long florescence lifetime and strong hydrophobic property. When the micelles formed, the pyrene molecule would transfer into the hydrophobic cores of the micelles from the aqueous solution, and a change of the photophysical property of pyrene could be observed. As the concentration of conjugates increased, red shifts of the maximum peak for pyrene could be observed, indicating the formation of micelles. The results showed that the maximum peak of the spectra shifted from 333 to 335 nm (data not shown). The pyrene fluorescence intensity ratios (I335/I333) were plotted against the logarithm of the conjugate concentration (Figure 4). And the inflection point of the curve is the CMC value of the conjugate. The CMC value of PF127 offered by BASF is 3.512 102 mg/ml. The detected
Pluronic-attached polyamidoamine dendrimers: drug delivery
DOI: 10.3109/03639045.2014.908899
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Figure 5. Hemolysis assay of unconjugated PAMAM dendrimer and PF127-attached PAMAM dendrimers. Results are expressed as mean ± SD (n ¼ 3).
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Figure 4. Plot of I335/I333 against the logarithm of PAMAM-n1 PF127 (A) and PAMAM-n2 PF127 (B) concentration. Table 2. Size and zeta potential of the dendrimers. Dendrimers
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CMC values of PAMAM-n1 PF127 and PAMAM-n2 PF127 are 0.1603 and 0.2214 mg/ml separately. The CMC values of the conjugates are much higher than the theoretical value of PF127. While the conjugates have similar CMC values. Previous studies showed that CMC values were influenced by both the hydrophobic blocks and the hydrophilic blocks26. The conjugation between PAMAM and PF127 changed the hydrophilic blocks of PF127, thus higher CMC values were observed. Size and zeta potential of the conjugates As shown in Table 2, the size of the dendrimers increased for the conjugation of PF127. And the size became bigger as the conjugation ratios increased. The detected mean size of PAMAM dendrimer (185.2 nm) is much larger than the PAMAM dendrimer itself (510 nm). According to previous study, the dramatical increase of the size could be caused by the aggregation of the PAMAM dendrimer27,28. And, the size of conjugates is bigger than 200 nm for the formation of micelles. The reduction of the number of amino groups on the surface led to a significant decrease of zeta potential. The results also showed that the zeta potential of the conjugates became lower as the conjugation ratios increased. The reduced zeta potential could help decrease the hemolytic toxicity and cytotoxicity of PAMAM dendrimer for the weaker interaction with the negatively charged cell surfaces29.
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Figure 6. In vitro cytotoxicity of unconjugated PAMAM dendrimer and PF127-attached PAMAM dendrimers in MCF-7 cells after application on cells for 48 h. Results are expressed as mean ± SD (n ¼ 3).
The release amount of hemoglobin was used to quantify the membrane-damaging properties of the dendrimers. As Figure 5 shown, unconjugated PAMAM showed 51.3% hemolysis at the concentration of 2.5 mg/ml. However, after the PF127 conjugation the hemolytic percentage of the dendrimers decreased dramatically to 51% (p50.001) at the concentration of 2.5 mg/ml. The hemolysis of unconjugated PAMAM reached 75.1% at the concentration of 5 mg/ml, while PAMAM-n1 PF127 showed 5.6% (p50.001) hemolysis and PAMAM-n2 PF127 showed 2.4% (p50.001) hemolysis. So the increased PF127 conjugation could help decrease the hemolytic percentage. The cell viabilities of PAMAM and PF127-attached PAMAM dendrimers were compared to evaluate their cytotoxicity. As shown in Figure 6, at the concentration of 180 mg/ml, PAMAM-n1 PF127 increased cell viability of PAMAM from 42.5% to 80.9% (p50.001) and PAMAM-n2 PF127 increased cell viability of PAMAM from 42.5% to 79.6% (p50.005) in MCF-7 cells. At the highest concentration of the dendrimers, the conjugates increased cell viability of PAMAM from 39.6% to 460% (p50.005). So the PF127 conjugation could decrease the cytotoxicity of PAMAM significantly. And the conjugates showed similar cytotoxicity. DOX loading
Toxicity of the conjugates The hemolysis and cytotoxicity assay were used to evaluate the toxicity of the PAMAM and conjugated PAMAM dendrimers. The results showed that the conjugation of PF127 could significantly reduce the toxicity of PAMAM dendrimer mainly for the reduced zeta potential.
The PF127-attached PAMAM dendrimers both have a hydrophobic interior and a hydrophilic surface. The hydrophobic interior makes the conjugates have the ability to encapsulate hydrophobic drugs, and the hydrophilic surface makes them biocompatible. DOX was chosen as a hydrophobic drug model for its wide use. DOX with a hydrophobic nature was
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PF127 chains. But as time went by, the blocking effect of PF127 chains decreased.
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Figure 7. In vitro DOX release from PF127-n-PAMAM dendrimer–DOX complexes in PBS (pH 7.4). Results are expressed as means ± SD from three independent experiments.
encapsulated into the hydrophobic interior of the conjugates. The amount of DOX was certain and excess compared to the conjugates. Conjugates of equivalent molar were added to form the PF127-attached PAMAM dendrimer–DOX complexes. The encapsulation numbers of DOX molecules per PAMAM-n1 PF127 and PAMAM-n2 PF127 conjugates are 12.87 and 4.07, respectively. The results show that more PF127 chains conjugated to the dendrimer could decrease the drug-loading capacity for the blocking effect of PF127. From another point of view, Kojima et al.16 found that the ability to encapsulate drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts with the same conjugation ratio, and the M-PEG(2000)-G4 PAMAM dendrimer had the highest ability to retain 6.5 DOX molecules. The results show that PAMAM-19% PF127 has higher ability to encapsulate DOX. Sustained release of DOX from DOX-loaded complexes To obtain the release behavior of DOX from the complexes, the DOX-loaded PF127-attached PAMAM dendrimers were dissolved in PBS and dialyzed against PBS (pH 7.4). The pure DOX was treated as the complexes. As Figure 7 shown, the release of DOX from PF127-attached PAMAM dendrimer–DOX complexes is much slower and lower than that of DOX solution. The cumulative release of DOX from the complexes showed that the drug was released in a sustained manner, while pure DOX was released quickly. The release behavior of DOX from the complexes followed a biphasic pattern. At first, it behaved a faster release, then followed a sustained release. Within 24 h, the cumulative release of DOX from the complexes was 530%, while almost 100% of pure DOX was released. So the hydrophobic interior of the conjugates is extremely useful for effective encapsulation and retention of the hydrophobic DOX drug. And this would help decrease the leakage of DOX in physiology situation. Compared the two complexes of different PF127 conjugation ratios, it showed different release behavior. In the first 2 h, cumulative release of DOX from the PAMAM-n1 PF127–DOX complex was 13% compared to 5% cumulative release of DOX from the PAMAM-n2 PF127–DOX complex. Similar result was got at the fourth hour. The former was 16% compared to 9% of the latter. However, the results of cumulative release of DOX from the complexes were similar within 24 h. The former was 25% compared to 24% of the latter. So in the first few hours, the rate of release would be slowed down as the conjugation ratio increased for the block of
In this study, we tried to prove whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems. A simple method was used to synthesize two conjugation ratios of PF127-attached PAMAM dendrimers successfully. And DOX was used as a hydrophobic drug model to form PF127-attached PAMAM dendrimer–DOX complexes. Firstly, the drug delivery system showed low hemolysis and cytotoxicity compared with unconjugated PAMAM dendrimer. Secondly, the conjugates showed high ability to encapsulate drugs. And it was demonstrated that the PAMAM-19% PF127 had the higher ability to retain 12.87 DOX molecules. Finally, the release of drugs from the drug-loaded system was in a sustained manner, and this would help decrease the leakage of drugs in physiology situation. Thus, the PF127-attached PAMAM dendrimers may be potential carriers for hydrophobic drug delivery.
Declaration of interest The authors gratefully acknowledge the Clinical Research Fund Project of Zhejiang Province Medical Association (Project No.2011ZYC-A95) for their financial support of this research.
References 1. Tomalia DA, Naylor AM, Goddard WA. Starburst dendrimers: molecular-level control of size, shape, surface chemistry, topology, and flexibility from atoms to macroscopic matter. Angew Chem Int Ed 1990;29:138–75. 2. Jang SH, Choi SJ, Oh JH, et al. Nonviral gene delivery to human ovarian cancer cells using arginine-grafted PAMAM dendrimer. Drug Dev Ind Pharm 2011;37:41–6. 3. Qi R, Gao Y, Tang Y, et al. PEG-conjugated PAMAM dendrimers mediate efficient intramuscular gene expression. AAPS J 2009;11: 395–405. 4. Hong S, Leroueil PR, Majoros IJ, et al. The binding avidity of a nanoparticle-based multivalent targeted drug delivery platform. Chem Biol 2007;14:107–15. 5. Jevprasesphant R, Penny J, Attwood D, et al. Engineering of dendrimer surfaces to enhance transepithelial transport and reduce cytotoxicity. Pharm Res 2003;20:1543–50. 6. Majoros IJ, Thomas TP, Mehta CB, Baker JR. Poly (amidoamine) dendrimer-based multifunctional engineered nanodevice for cancer therapy. J Med Chem 2005;48:5892–9. 7. Ma K, Hu MX, Qi Y, et al. PAMAM–Triamcinolone acetonide conjugate as a nucleus-targeting gene carrier for enhanced transfer activity. Biomaterials 2009;30:6109–18. 8. Arima H, Yoshimatsu A, Ikeda H, et al. Folate-pegappended dendrimer conjugate with a-cyclodextrin as a novel cancer cell-selective siRNA delivery carrier. Mol Pharm 2012;9: 2591–604. 9. Sideratou Z, Tsiourvas D, Paleos CM. Solubilization and release properties of PEGylated diaminobutane poly (propylene imine) dendrimers. J Colloid Interface Sci 2001;242:272–6. 10. Bhadra D, Bhadra S, Jain S, Jain N. A PEGylated dendritic nanoparticulate carrier of fluorouracil. Int J Pharm 2003;257: 111–24. 11. Jiang YY, Tang GT, Zhang LH, et al. PEGylated PAMAM dendrimers as a potential drug delivery carrier: in vitro and in vivo comparative evaluation of covalently conjugated drug and noncovalent drug inclusion complex. J Drug Target 2010;18: 389–403. 12. Zhu S, Hong M, Zhang L, et al. PEGylated PAMAM dendrimerdoxorubicin conjugates: in vitro evaluation and in vivo tumor accumulation. Pharm Res 2010;27:161–74. 13. Chandrasekar D, Sistla R, Ahmad FJ, et al. Folate coupled poly (ethyleneglycol) conjugates of anionic poly (amidoamine) dendrimer for inflammatory tissue specific drug delivery. J Biomed Mater Res A 2007;82:92–103.
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14. Ly TU, Tran NQ, Hoang TKD, et al. Pegylated dendrimer and its effect in fluorouracil loading and release for enhancing antitumor activity. J Biomed Nanotechnol 2013;9:213–20. 15. Zhu S, Qian L, Hong M, et al. RGD-modified PEG-PAMAM-DOX conjugate: in vitro and in vivo targeting to both tumor neovascular endothelial cells and tumor cells. Adv Mater 2011;23:H84–9. 16. Kojima C, Kono K, Maruyama K, Takagishi T. Synthesis of polyamidoamine dendrimers having poly (ethylene glycol) grafts and their ability to encapsulate anticancer drugs. Bioconjugate Chem 2000;11:910–17. 17. Jeon E, Kim HD, Kim JS. Pluronic-grafted poly-(L)-lysine as a new synthetic gene carrier. J Biomed Mater Res A 2003;66:854–9. 18. Alexander S, de Vos WM, Castle TC, et al. Growth and shrinkage of pluronic micelles by uptake and release of flurbiprofen: variation of pH. Langmuir 2012;28:6539–45. 19. Gao M, Xu H, Zhang C, et al. Preparation and characterization of curcumin thermosensitive hydrogels for intratumoral injection treatment. Drug Dev Ind Pharm 2013. [Epub ahead of print]. DOI: 10.3109/03639045.2013.838579. 20. Li N, Yang X, Zhai G, Li L. Multifunctional pluronic/poly (ethylenimine) nanoparticles for anticancer drug. J Colloid Interface Sci 2010;350:117–25. 21. Zhang W, Shi Y, Chen Y, et al. Enhanced antitumor efficacy by paclitaxel-loaded pluronic P123/F127 mixed micelles against nonsmall cell lung cancer based on passive tumor targeting and modulation of drug resistance. Eur J Pharm Biopharm 2010;75: 341–53.
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22. Wei Z, Hao J, Yuan S, et al. Paclitaxel-loaded Pluronic P123/F127 mixed polymeric micelles: formulation, optimization and in vitro characterization. Int J Pharm 2009;376:176–85. 23. Kabanov AV, Batrakova EV, Yu Alakhov V. An essential relationship between ATP depletion and chemosensitizing activity of PluronicÕ block copolymers. J Control Rel 2003;91:75–83. 24. Wang Y, Yu L, Han L, et al. Difunctional Pluronic copolymer micelles for paclitaxel delivery: synergistic effect of folate-mediated targeting and Pluronic-mediated overcoming multidrug resistance in tumor cell lines. Int J Pharm 2007;337:63–73. 25. Hedden RC, Bauer BJ. Structure and dimensions of PAMAM/PEG dendrimer-star polymers. Macromolecules 2003;36:1829–35. 26. Sezgin Z, Yu¨ksel N, Baykara T. Preparation and characterization of polymeric micelles for solubilization of poorly soluble anticancer drugs. Eur J Pharm Biopharm 2006;64:261–8. 27. Santos JL, Oliveira H, Pandita D, et al. Functionalization of poly (amidoamine) dendrimers with hydrophobic chains for improved gene delivery in mesenchymal stem cells. J Control Rel 2010;144: 55–64. 28. Han M, Lv Q, Tang X-J, et al. Overcoming drug resistance of MCF7/ADR cells by altering intracellular distribution of doxorubicin via MVP knockdown with a novel siRNA polyamidoamine-hyaluronic acid complex. J Control Rel 2012;163:136–44. 29. Halets I, Shcharbin D, Klajnert B, Bryszewska M. Contribution of hydrophobicity, DNA and proteins to the cytotoxicity of cationic PAMAM dendrimers. Int J Pharm 2013;454:1–3.
Erratum The concentration unit in Figure 6 has been corrected from mgml1 to mgml1 since the first online publication.
RESEARCH ARTICLE
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Preparation and in vitro characterization of pluronic-attached polyamidoamine dendrimers for drug delivery Zhuojun Gu1*, Meng Wang2*, Qiongyan Fang1, Huaiyu Zheng1, Feiyue Wu1, Dai Lin1, Ying Xu1, and Yi Jin2 1
Zhoushan Hospital, Zhoushan, P.R. China and 2College of Pharmaceutical Sciences, Institute of Pharmaceutics, Zhejiang University, Hangzhou, P.R. China Abstract
Keywords
Context: Polyamidoamine (PAMAM) dendrimers have attracted lots of interest as drug carriers. And little study about whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems has been carried on. Objective: Pluronic F127 (PF127) attached PAMAM dendrimers were designed as novel drug carriers. Methods: Two conjugation ratios of PF127-attached PAMAM dendrimers were synthesized. 1H nuclear magnetic resonance (1H-NMR), Fourier transform infrared spectrum (FTIR), element analysis and ninhydrin assay were used to characterize the conjugates. Size, zeta potential and critical micelle concentrations (CMC) were also detected. And DOX was incorporated into the hydrophobic interior of the conjugates. Studies on their drug loading and drug release were carried on. Furthermore, hemolysis and cytotoxicity assay were used to evaluate the toxicity of the conjugates. Results and discussion: PF127 was successfully conjugated to the fifth generation PAMAM dendrimer at two molar ratios of 19% and 57% (PF127 to surface amine per PAMAM dendrimer molecular). The conjugates showed an increased size and a reduced zeta potential. And higher CMC values were obtained than pure PF127. Compared with unconjugated PAMAM dendrimer, PF127 conjugation significantly reduced the hemolytic toxicity and cytotoxicity of PAMAM dendrimer in vitro. The encapsulation results showed that the ability to encapsulate DOX by the conjugate of 19% conjugation ratio was better than that of 57% conjugation ratio. And the maximum is 12.87 DOX molecules per conjugate molecule. Moreover, the complexes showed a sustained release behavior compared to pure DOX. Conclusion: Findings from the in vitro study show that the PF127-attached PAMAM dendrimers may be potential carriers for drug delivery.
Conjugate, DOX, drug delivery, PAMAM dendrimer, pluronic F127, toxicity
Introduction Dendrimers are unique synthetic macromolecules with highly branched structure and globular shape1. These have definite molecular weight, shape, size and host–guest entrapment properties, which make them be ideal carriers for drugs and genes. Poly(amidoamine) (PAMAM) dendrimers have been widely studied and used among the dendrimers so far. The PAMAM dendrimers could be modified with multiple functionalities such as drugs, genes, targeting molecules and imaging agents for the plentiful surface groups2–6. In our previous study, PAMAM– triamcinolone acetonide (PAMAM–TA) conjugates were synthesized for efficient translocation of pDNA into the nucleus7.
*These two authors contributed equally to this work. Address for correspondence: Yi Jin, College of Pharmaceutical Sciences, Institute of Pharmaceutics, Zhejiang University, Hangzhou 310058, P.R. China. Tel/Fax: +86 571 88208435. E-mail: [email protected] Zhuojun Gu, Zhoushan Hospital, Zhoushan 316000, P.R. China. Tel/ Fax: +86 580 2292847. E-mail: [email protected]
History Received 19 June 2013 Revised 24 October 2013 Accepted 9 March 2014 Published online 18 April 2014
And the conjugates showed high transfection efficiency and low toxicity. Many PEGylated dendrimeric systems have been explored as potential drug delivery agents at present8,9. The PEG conjugation can increase drug loading and overcome a few drawbacks like hemolytic toxicity, drug leakage and so on10. In the previous studies, PEGylated PAMAM dendrimers can load drugs by two ways. One way is by conjugation with drugs through covallent bond11,12. And another way is by physical encapsulation with drugs13,14. For example, Zhu et al.15 prepared RGD-modified PEGPAMAM-DOX conjugate for targeted cancer therapies. And, Kojima et al.16 prepared M-PEG-attached PAMAM dendrimers to encapsulate DOX by the hydrophobic interior or MTX by the basic interior. While little study about whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems has been carried on. So the conjugation of PAMAM by pluronic for drug delivery would be an attractive field to research. Pluronic is a kind of polymer surfactants, which has a triblock structure: PEO-PPO-PEO. Pluronic is amphiphilic for the hydrophilic poly(ethylene oxide) (PEO) blocks and the hydrophobic poly(propylene oxide) (PPO) blocks. Pluronic has the ability to
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interact with biological membranes and to enhance transport of compounds into the cells17. And, it has been widely used in micelles, nanoparticles, hydrogels and so on18–21. Pluronic F127 (PF127) was used for its biocompatibility and its approval by FDA22. And DOX-loaded L61/F127 micelles were evaluated in Phase II study in 200223. In our present work, two conjugation ratios of PF127-attached PAMAM dendrimers were synthesized. Furthermore, DOX was chosen as a hydrophobic model drug and incorporated into the hydrophobic interior of the conjugates. Studies on their toxicity, drug loading and drug release were carried on to evaluate whether the conjugates could be potential drug carriers. The new drug carriers were expected to incorporate much more DOX and show better release behavior with decreased toxicity.
Materials and methods Materials Pluronic F127 was kindly supplied by BASF China Ltd. (Shanghai, China). The fifth generation PAMAM dendrimer was purchased from Weihai City Chenyuan Silicone New Material Co., Ltd. (Weihai, China). 4-Nitrophenyl chloroformate and 3-(4, 5-dimethylthi-zaol-2-yl)-2, 5-diphenyltetrazolum bromide (MTT) were purchased from Sigma–Aldrich China Inc. (Shanghai, China). Doxorubicin hydrochloride (DOXHCl) was purchased from Haikou Manfanyuan Chemical Co., Ltd. (Haikou, China). Fetal bovine serum (FBS) was purchased from Sijiqing Biologic Co., Ltd. (Hangzhou, China). RPMI1640 medium (RPMI), trypsin-EDTA solution (0.5% trypsin, 5.3 Mm EDTA tetrasodium) and PBS (phosphate-buffered saline) were purchased from Jinuo Biomedical Technology Co., Ltd (Hangzhou, China). Distilled water used in the synthesis was prepared by Milli-Q (Millipore, Billerica, MA). All other chemicals were analytical grade. Activation of pluronic F127 Pluronic F127 (PF127) was activated with 4-nitrophenyl chloroformate as described earlier24. 4-Nitrophenyl chloroformate (0.4 mmol) was dissolved in benzene and then added to PF127 (0.4 mmol) dissolved in benzene drop by drop. Then the reaction mixture was stirred for 24 h at room temperature under a nitrogen atmosphere. After reaction, the activated PF127 was precipitated three times using 8-fold excess of petroleum ether (bp 30–60 C) to remove the unreacted 4-nitrophenyl chloroformate. By centrifugation, the activated PF127 was recovered and kept under vacuum overnight to remove the remaining solvent. The product was analyzed with 1H nuclear magnetic resonance (NMR) (Bruker AVIII500M, Fa¨llanden, Switzerland) and Fourier transform infrared spectrum (FTIR) (JASCO FT/IR4100, Tokyo, Japan). D2O was purchased from Aladdin (Shanghai, China) as a NMR solvent. Synthesis of PF127-attached PAMAM dendrimers About 1.3 mmol of the fifth generation PAMAM dendrimer was dissolved in dimethyl sulfoxide. The activated PF127 was added into the solution at different molar ratios (25% and 65% per PAMAM, respectively). Then the solution was stirred to react for 4 or 6 days at room temperature (depending on the PF127 modification ratio). After reaction, the solution was dialyzed against distilled water for 3 or more days. Finally, the PF127attached PAMAM dendrimers were obtained by lyophilization. The products were analyzed with 1H-NMR and FTIR. Element analysis (ThermoFinnigan Flash EA 1112, Milan, Italy) was measured to determine the exact number of the pluronic chains conjugated to the PAMAM dendrimer. The average size and zeta
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potential values were measured with a Zetasizer (Malvern NANO ZS90, Worcestershire, UK). Ninhydrin assay Ninhydrin assay was used to determine the primary amine contents of the conjugates. The ninhydrin reagent was prepared on the day of the assay and used in 24 h. About 0.4 g ninhydrin and 0.06 g hydrindantin were dissolved in 15 ml of dimethyl sulfoxide followed by adding 5 ml of sodium acetate buffer (4 M) while flushing with nitrogen. The reagent was stored in 4 C. About 0.5 ml of the reagent was added to 0.5 ml of the sample. The tubes were capped immediately, then shaken briefly by hand and heated in a boiling water bath for 30 min. When the tubes were cooled down, 9 ml of 50% alcohol was added to each tube and the solutions were mixed with a vortex mixer for 15 s. The absorbance of each solution was measured with a UV-Vis spectrophotometer (Persee TU-1800PC, Beijing, China) at 570 nm. CMC of the PF127-attached PAMAM dendrimers Critical micelle concentrations (CMC) of the conjugates were determined to prove the potential of micelle formation. Fluorescence measurements were carried out by using pyrene as a probe to determine the CMC. Pyrene in acetone was added to a series of volumetric flasks, and then the solvent was evaporated. Different volumes of the conjugates were added to the volumetric flasks and diluted with distilled water separately. The final concentration of pyrene was 6.0 107 M, and the concentrations of the conjugates varied from 1.0 106 to 1.0 mg/ml. The solutions were incubated at 37 C with mild shake overnight to equilibrate the pyrene and the conjugates, and finally the solutions were incubated at room temperature. Fluorescence spectra were measured by a fluorescence spectrophotometer (JASCO FP 6500, Tokyo, Japan). Excitation spectra were obtained from the scanning excitation spectra from 300 to 360 nm, fixing the emission wavelength at 390 nm. Encapsulation of DOX by PF127-attached PAMAM dendrimers About 3.2 mg DOXHCl was dissolved in 1.5 ml methanol: acetone (1:1), and 5 ml triethylamine was further added to transform DOXHCl into hydrophobic DOX (2 mg/ml). PF127attached PAMAM dendrimers of equivalent molar were dissolved in PBS (pH 7.4), and then mixed with the same volume of the above DOX solution separately. The DOX was excess compared to the conjugates. The mixed solution was stirred for 24 h to evaporate the organic solvent. Then the solution was centrifuged at 13 000 g for 10 min to remove the unloaded DOX. Appropriate amount of the supernatant was diluted with PBS (pH 7.4) and absorbance was measured to determine the content of DOX with a UV-Vis spectrophotometer at 479 nm. Finally the supernatant was lyophilized to get the PF127-attached PAMAM dendrimer–DOX complexes. Release of DOX from PF127-attached PAMAM dendrimer–DOX complexes PF127-attached PAMAM dendrimer–DOX complexes were dispersed in 2 ml PBS (pH 7.4) separately before transferred into the dialysis bag (MWCO ¼ 4000). Then each bag was placed in 20 ml PBS (pH 7.4), and shaken at a rate of 100 g at 37 C. At the predetermined time intervals, 2 ml of the release medium was taken out, and 2 ml of fresh medium was added after each collection. The absorption of the release medium was measured by a UV-Vis spectrophotometer at 479 nm.
DOI: 10.3109/03639045.2014.908899
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In vitro hemolysis assay The hemolytic activity of the PAMAM and conjugated PAMAM dendrimers was investigated. Blood of New Zealand white rabbit was collected in heparinized tubes and centrifuged at 3000 g for 10 min. The cell pellet was washed three times with cold PBS (pH 7.4) by centrifugation at 3000 g for 10 min each time and finally resuspended in PBS (pH 7.4) to reach a concentration of 1.5% (v/v). The suspension of red blood cells was freshly prepared and used within 24 h after collection. Solutions of PAMAM dendrimer and PF127-attached PAMAM dendrimers were prepared in PBS (pH 7.4) and diluted to different concentrations. The polymer solutions were mixed with equal volume of the erythrocytes separately. The mixtures were incubated at 37 C for 4 h and centrifuged at 13 000 g for 10 min. Absorption of the supernatant was measured by a UV-Vis spectrophotometer at 540 nm to determine the release of hemoglobin. About 0.2% Triton X-100 was regarded as the positive control and PBS (pH 7.4) was regarded as the negative control.
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In vitro cytotoxicity assay The cytotoxicity of the PAMAM and PF127-attached PAMAM dendrimers was evaluated by MTT assay. MCF-7 cells were seeded into a 96-well plate at a density of 5 103 cells per well in 100 ml of culture medium and allowed to attach for 24 h. Then the cells were incubated with 25 ml of various determined concentrations of conjugates. After 48-h incubation, 31.5 ml of MTT solution (5 mg/ml in PBS) was added to every well and the cells were incubated for another 4 h. Then the culture medium was completely removed and 200 ml DMSO was added. Absorbance of each well was measured with a Microplate Reader (Bio-Rad, Hercules, CA) at 570 nm.
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Statistical analysis Data which were expressed as mean ± SD were repeated at least three times. Statistical significance was determined using Student’s t-test.
Results and discussion Synthesis and characterization of activated PF127 and PF127-attached PAMAM dendrimers PF127 was activated with equimolar amount of 4-nitrophenyl chloroformate to synthesis PF127 with the single hydroxyl end activated. So the number of both hydroxyl ends of PF127 activated was minimized. The characterization of the activated PF127 and PF127-attached PAMAM dendrimers was determined by 1H-NMR in D2O (Figure 1). The degree of activation was 81.35% from the peak intensity ratio of the aryl protons of the nitrophenyl groups (NO2–C6H4–, d ¼ 6.9–8.4 ppm) to the methyl protons of PF127 (–CH3, d ¼ 1.19 ppm) (Figure 1B). The peak in PF127-attached PAMAM dendrimers at 1.19 ppm corresponds to the methyl group (–CH3) of the PF127 (Figure 1A). And peaks belonging to PAMAM (Figure 1C) at 2.46 (–NCH2CH2CO–), 2.63 (–CONHCH2CH2N–), 2.76 (–CONHCH2CH2N), 2.83 (–NCH2CH2CO–), 3.27 and 3.37 (–CONHCH2CH2NH2, NCH2CH2NHCO–), are still observed in the conjugates (Figure 1D and E), which confirmed the formation of conjugation. A little shift of the peaks was shown in the spectra because of the conjugation of PF127 to the PAMAM dendrimer. No peaks of the aryl protons of the nitrophenyl groups (NO2–C6H4–, d ¼ 6.9-8.4 ppm) were observed in the spectra of the conjugates. So pure conjugates were got without activated PF127 (APF127) left.
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Figure 1. 1H nuclear magnetic resonance (NMR) spectra of PF127 (A), APF127 (B), PAMAM (C), PAMAM-n1 PF127 (D) and PAMAM-n2 PF127 (E).
FTIR was also used to characterize the activated PF127 and PF127-attached PAMAM dendrimers. FTIR spectra were recorded in the range between 4000 and 400 cm1 as shown in Figure 2. New absorption at 1770 cm1 belonging to the stretching vibration frequency of C ¼ O of eater group was shown in the spectrum of activated PF127. So the PF127 was successfully activated. Compared the FTIR spectra of the conjugates with that of PF127, new absorptions at 1649
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Figure 2. FTIR spectra of PF127, APF127, PAMAM-n1 PF127 and PAMAM-n2 PF127. Table 1. PF127 conjugation ratios of PF127-attached PAMAM dendrimers determined by element analysis.
Products PAMAM-n1 PF 127 PAMAM-n2 PF127
Theoretical PF127 conjugation ratios (%)
Detected carbon (%)
Detected nitrogen (%)
The theoretical number of PF127 chains
The detected number of PF127 chains
Detected PF127 conjugation ratios (%)
25% 65%
55.53 56.65
2.09 0.75
32.00 83.20
24.28 72.94
18.97 56.98
and 1555 cm1 were shown in the spectra of the conjugates, and absorption at 1770 cm1 was not observed. Absorption at 1649 cm1 belongs to the stretching vibration frequency of C ¼ O of secondary amide group, and absorption at 1555 cm1 belongs to the stretching vibration frequency of C–N and the bending vibration of NH of secondary amide group of the products. So the same conclusion could be drawn that the conjugates were successfully synthesized without APF127 left. Element analysis was used to analyse the ratio of C, N and H elements of the final products and the results were listed in Table 1. The exact number of the PF127 chains conjugated to the PAMAM dendrimer was got through the C/N ratio. The added APF127 was not fully conjugated to the PAMAM dendrimer. Even after a long reaction time, PAMAM-25% PF127 achieved a PF127 conjugation ratio of 19%, and PAMAM-65% PF127 achieved a PF127 conjugation ratio of 57%. One of the reasons is that a further reaction between the APF127 and the primary amine groups of PAMAM dendrimer could be blocked because of the long and convoluted chain of PF127. And similar results were achieved in the process of PEG conjugation to PAMAM dendrimers3,25. PAMAM-n1 PF127 and PAMAM-n2 PF127 were used to represent the final products (n represents the conjugation ratio, and n15n2). Determination of primary amines The absorption intensity of the solution is proportional to the amount of free amines. The primary amine contents of the dendrimers were determined by ninhydrin assay. As Figure 3 shown, much lower absorption intensity of the PF127-attached PAMAM dendrimers than PAMAM dendrimer was observed at the same concentration. The primary amine density of PAMAM decreased for the conjugation of PF127 to the PAMAM dendrimer. As for the different conjugation ratios of the conjugates, the absorption decreased as the conjugation ratios increased. The absorption intensity of PAMAM-n2 PF127 was lower than that of PAMAM-n1 PF127, so a conclusion could be drawn that the value of n2 is bigger than n1. Thus the different
PAMAM PAMAM-n1 PF127 PAMAM-n2 PF127
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Figure 3. UV curves of PAMAM, PAMAM-n1 PF127 and PAMAM-n2 PF127 for ninhydrin assay.
absorption intensity of the conjugates could reflect the degree of conjugation. CMC of the PF127-attached PAMAM dendrimers PF127 is amphiphilic for its PEO-PPO-PEO structure. It has the ability to form micelles in aqueous solutions above the critical micelle concentration. To demonstrate that the PF127-attached PAMAM dendrimers could also form micelles above the CMC, a fluorescent measurement was taken by using pyrene as a probe. Pyrene has long florescence lifetime and strong hydrophobic property. When the micelles formed, the pyrene molecule would transfer into the hydrophobic cores of the micelles from the aqueous solution, and a change of the photophysical property of pyrene could be observed. As the concentration of conjugates increased, red shifts of the maximum peak for pyrene could be observed, indicating the formation of micelles. The results showed that the maximum peak of the spectra shifted from 333 to 335 nm (data not shown). The pyrene fluorescence intensity ratios (I335/I333) were plotted against the logarithm of the conjugate concentration (Figure 4). And the inflection point of the curve is the CMC value of the conjugate. The CMC value of PF127 offered by BASF is 3.512 102 mg/ml. The detected
Pluronic-attached polyamidoamine dendrimers: drug delivery
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Figure 5. Hemolysis assay of unconjugated PAMAM dendrimer and PF127-attached PAMAM dendrimers. Results are expressed as mean ± SD (n ¼ 3).
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Figure 4. Plot of I335/I333 against the logarithm of PAMAM-n1 PF127 (A) and PAMAM-n2 PF127 (B) concentration. Table 2. Size and zeta potential of the dendrimers. Dendrimers
80 60 40 20
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Zeta potential (mV)
185.2 254.6 339.7
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Cell viability/%
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CMC values of PAMAM-n1 PF127 and PAMAM-n2 PF127 are 0.1603 and 0.2214 mg/ml separately. The CMC values of the conjugates are much higher than the theoretical value of PF127. While the conjugates have similar CMC values. Previous studies showed that CMC values were influenced by both the hydrophobic blocks and the hydrophilic blocks26. The conjugation between PAMAM and PF127 changed the hydrophilic blocks of PF127, thus higher CMC values were observed. Size and zeta potential of the conjugates As shown in Table 2, the size of the dendrimers increased for the conjugation of PF127. And the size became bigger as the conjugation ratios increased. The detected mean size of PAMAM dendrimer (185.2 nm) is much larger than the PAMAM dendrimer itself (510 nm). According to previous study, the dramatical increase of the size could be caused by the aggregation of the PAMAM dendrimer27,28. And, the size of conjugates is bigger than 200 nm for the formation of micelles. The reduction of the number of amino groups on the surface led to a significant decrease of zeta potential. The results also showed that the zeta potential of the conjugates became lower as the conjugation ratios increased. The reduced zeta potential could help decrease the hemolytic toxicity and cytotoxicity of PAMAM dendrimer for the weaker interaction with the negatively charged cell surfaces29.
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Figure 6. In vitro cytotoxicity of unconjugated PAMAM dendrimer and PF127-attached PAMAM dendrimers in MCF-7 cells after application on cells for 48 h. Results are expressed as mean ± SD (n ¼ 3).
The release amount of hemoglobin was used to quantify the membrane-damaging properties of the dendrimers. As Figure 5 shown, unconjugated PAMAM showed 51.3% hemolysis at the concentration of 2.5 mg/ml. However, after the PF127 conjugation the hemolytic percentage of the dendrimers decreased dramatically to 51% (p50.001) at the concentration of 2.5 mg/ml. The hemolysis of unconjugated PAMAM reached 75.1% at the concentration of 5 mg/ml, while PAMAM-n1 PF127 showed 5.6% (p50.001) hemolysis and PAMAM-n2 PF127 showed 2.4% (p50.001) hemolysis. So the increased PF127 conjugation could help decrease the hemolytic percentage. The cell viabilities of PAMAM and PF127-attached PAMAM dendrimers were compared to evaluate their cytotoxicity. As shown in Figure 6, at the concentration of 180 mg/ml, PAMAM-n1 PF127 increased cell viability of PAMAM from 42.5% to 80.9% (p50.001) and PAMAM-n2 PF127 increased cell viability of PAMAM from 42.5% to 79.6% (p50.005) in MCF-7 cells. At the highest concentration of the dendrimers, the conjugates increased cell viability of PAMAM from 39.6% to 460% (p50.005). So the PF127 conjugation could decrease the cytotoxicity of PAMAM significantly. And the conjugates showed similar cytotoxicity. DOX loading
Toxicity of the conjugates The hemolysis and cytotoxicity assay were used to evaluate the toxicity of the PAMAM and conjugated PAMAM dendrimers. The results showed that the conjugation of PF127 could significantly reduce the toxicity of PAMAM dendrimer mainly for the reduced zeta potential.
The PF127-attached PAMAM dendrimers both have a hydrophobic interior and a hydrophilic surface. The hydrophobic interior makes the conjugates have the ability to encapsulate hydrophobic drugs, and the hydrophilic surface makes them biocompatible. DOX was chosen as a hydrophobic drug model for its wide use. DOX with a hydrophobic nature was
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PF127 chains. But as time went by, the blocking effect of PF127 chains decreased.
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Figure 7. In vitro DOX release from PF127-n-PAMAM dendrimer–DOX complexes in PBS (pH 7.4). Results are expressed as means ± SD from three independent experiments.
encapsulated into the hydrophobic interior of the conjugates. The amount of DOX was certain and excess compared to the conjugates. Conjugates of equivalent molar were added to form the PF127-attached PAMAM dendrimer–DOX complexes. The encapsulation numbers of DOX molecules per PAMAM-n1 PF127 and PAMAM-n2 PF127 conjugates are 12.87 and 4.07, respectively. The results show that more PF127 chains conjugated to the dendrimer could decrease the drug-loading capacity for the blocking effect of PF127. From another point of view, Kojima et al.16 found that the ability to encapsulate drugs increased with increasing dendrimer generation and chain length of poly(ethylene glycol) grafts with the same conjugation ratio, and the M-PEG(2000)-G4 PAMAM dendrimer had the highest ability to retain 6.5 DOX molecules. The results show that PAMAM-19% PF127 has higher ability to encapsulate DOX. Sustained release of DOX from DOX-loaded complexes To obtain the release behavior of DOX from the complexes, the DOX-loaded PF127-attached PAMAM dendrimers were dissolved in PBS and dialyzed against PBS (pH 7.4). The pure DOX was treated as the complexes. As Figure 7 shown, the release of DOX from PF127-attached PAMAM dendrimer–DOX complexes is much slower and lower than that of DOX solution. The cumulative release of DOX from the complexes showed that the drug was released in a sustained manner, while pure DOX was released quickly. The release behavior of DOX from the complexes followed a biphasic pattern. At first, it behaved a faster release, then followed a sustained release. Within 24 h, the cumulative release of DOX from the complexes was 530%, while almost 100% of pure DOX was released. So the hydrophobic interior of the conjugates is extremely useful for effective encapsulation and retention of the hydrophobic DOX drug. And this would help decrease the leakage of DOX in physiology situation. Compared the two complexes of different PF127 conjugation ratios, it showed different release behavior. In the first 2 h, cumulative release of DOX from the PAMAM-n1 PF127–DOX complex was 13% compared to 5% cumulative release of DOX from the PAMAM-n2 PF127–DOX complex. Similar result was got at the fourth hour. The former was 16% compared to 9% of the latter. However, the results of cumulative release of DOX from the complexes were similar within 24 h. The former was 25% compared to 24% of the latter. So in the first few hours, the rate of release would be slowed down as the conjugation ratio increased for the block of
In this study, we tried to prove whether pluronic-attached PAMAM dendrimers could be potential drug delivery systems. A simple method was used to synthesize two conjugation ratios of PF127-attached PAMAM dendrimers successfully. And DOX was used as a hydrophobic drug model to form PF127-attached PAMAM dendrimer–DOX complexes. Firstly, the drug delivery system showed low hemolysis and cytotoxicity compared with unconjugated PAMAM dendrimer. Secondly, the conjugates showed high ability to encapsulate drugs. And it was demonstrated that the PAMAM-19% PF127 had the higher ability to retain 12.87 DOX molecules. Finally, the release of drugs from the drug-loaded system was in a sustained manner, and this would help decrease the leakage of drugs in physiology situation. Thus, the PF127-attached PAMAM dendrimers may be potential carriers for hydrophobic drug delivery.
Declaration of interest The authors gratefully acknowledge the Clinical Research Fund Project of Zhejiang Province Medical Association (Project No.2011ZYC-A95) for their financial support of this research.
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Erratum The concentration unit in Figure 6 has been corrected from mgml1 to mgml1 since the first online publication.
