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Charge-Conversional and pH-Sensitive PEGylated Polymeric Micelles as Efficient Nanocarriers for Drug Deliverya Gong-Yan Liu, Min Li, Cong-Shan Zhu, Qiao Jin, Zong-Cai Zhang,* Jian Ji*
A novel amphiphilic copolymer, poly (ethylene glycol)-graft-polyethyleneimine/amide (PEGg-PEI/amide), is synthesized by grafting PEG and1,2-cis-Cyclohexanedicarboxylic anhydride onto the PEI. PEGylated polymeric micelles can be assembled from the amphiphilic copolymers with well-defined nano-sizes, and anticancer drugs are successfully loaded into micelle core formed by the amide. The amides with neighboring carboxylic acid groups exhibit pH-dependent hydrolysis and can reversibly shield the cationic charge of amine groups on the PEI, giving the micelles a charge-conversion property from negative to positive in acidic tumor tissue environment. Meanwhile, the cleavage of amide bonds at acidic pH also results in the disassembly of the micelle and pH-responsive drug release. These micelles are promising drug delivery systems due to their smart properties: PEGylation, suitable size, charge-conversion, and simultaneous pHsensitive drug release.
1. Introduction Dr. G.-Y. Liu, M. Li, Prof. Z.-C. Zhang Department of Biomass Chemistry and Engineering, National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, China E-mail: [email protected] C.-S. Zhu, Q. Jin, Prof. J. Ji MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China E-mail: [email protected] Supporting Information is available online from theOnline Library or from the author.
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Over the years, polymeric micelles as nano-scale drug delivery systems have shown great potential for delivering high payloads of therapeutic drugs to tumors.[1–6] Drugloaded polymeric micelles could control the time-dependent drug distribution in the body for improved therapeutic benefits and also reduced adverse effects.[7,8] However, for successful cancer treatment, drug carriers must overcome several transport barriers from systemic administration to access target sites, for example, rapid filtration in the kidney and clearance via the reticulo-endothelial system (RES) as well as transport from the bloodstream into target tissues or cells with controlled drug release.[9–13] Therefore, polymeric
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DOI: 10.1002/mabi.201400162
Charge-Conversional and pH-Sensitive PEGylated Polymeric . . . www.mbs-journal.de
micelles are supposed to be designed with smart properties, such as, reduced inherent toxicity, longevity in blood circulation, tissue accumulation and penetration, and stimuli-responsive drug release.[14–18] To address the renal excretion from kidney and capture by the RES, polymeric micelles with several tens nanometer and biocompatible PEG shell have been developed.[19–22] Through the systemic injection into blood circulation, the nano-scale size (10–100 nm) of micelles can effectively avoid rapid renal excretion and also allow micelles to accumulate into the tumor tissues by the enhanced permeability and retention (EPR) effect.[23] While the ‘‘stealth’’ property of PEGylated micelle will minimize the non-specific interactions with biological components including entrapment by RES, leading to prolonged blood circulation, enhanced passive tumor accumulation, and reduced the toxicity of the drug-loaded micelles.[12] After the accumulation at the target tissue with an acidic pH environment (pH 6.0),[24] micelles must furthermore cross the plasma membrane barriers into cancer cells and enable anti-cancer drugs to be released in a controlled manner. It has been reported that nanoparticles with a positive charged surface showed higher affinity for negative charged cell membranes and thus can be readily internalized by cells.[25] However, the positively charged surface may induce nonspecific interactions with serum, causing high cytotoxicity and excessive immune responsive.[26] On the contrary, negatively charged drug carriers have shown potential for protein resistance and exhibited prolonged circulation time for in vivo application.[27] Therefore, much effort has been concentrated on the development of surface charge-conversion methods triggered by the pH biosignals for drug delivery.[28–33] Recently, Kataoka and co-workers have proposed novel strategy for site-specific protein and gene release from smart polyion complex (PIC) micelles with a charge-conversion property from negative to positive in late endosomal/lysosomal acidic environments.[28–30] Shen et al. have also demonstrated a negative-to-positive charge-reversal technique for preparing polymeric micelles and conjugates for nuclear drug delivery, which could effectively enhance the cellular uptake of the nanoparticles and thereafter direct the carriers to localize in the nucleus.[31,32] Latter, Wang’s group has developed a tumor pH-responsive charge-conversional nanogel for promoted tumoral-cell uptake and Doxorubicin (DOX) delivery.[33] Despite the advantages of charge-conversion polymeric micelles, the simultaneously site-specific release of payload from the carrier is still one of the major obstacles in the development of an ideal drug delivery system. The capabilities of surface charge-conversion and simultaneous drug release in the target site can only be obtained by a smart delivery system that can respond to the change of biosignals, such as pH. In this study, we designed a
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PEGylated polymeric micelle with pH-sensitivity and simultaneous charge-conversion property for efficient anti-cancer drug delivery. PEI with high amine density was chose as the polycations due to its ‘‘proton-sponge’’ effect for tissue penetration.[34] Then, PEGylation was carried out by grafting PEG onto the PEI chain to increase the biocompatibility and reduce the toxicity of PEI. The PEG-gPEI copolymer was furthermore modified by reacting with 1,2-cis-Cyclohexanedicarboxylic anhydride to form PEG-gPEI/amide amphiphilic copolymers, which can self-assembled into polymeric micelles in aqueous solution. The amide bond obtained between the amine groups and anhydride had anionic charges with neighboring carboxylic acid groups, shielding the cationic charge of amine groups on the PEI. The resulting amide was stable at neutral pH, but would be cleaved at tumor pH[31] to revert back the initial amine groups and disassemble the formed micelles with the fast payload release. Therefore, modification with 1,2-cisCyclohexanedicarboxylic anhydride resulted in polymeric micelles having charge-conversional and pH-sensitive properties in tumor tissue acidic environments. After drug loading, such kind of PEGylated polymeric micelles with several tens of nanometer and negative charge surface would possess longevity in blood circulation and achieve successful tissue accumulation via EPR effect. In the following stage, the micelles could be activated to be positively charged for enhanced cellular internalization and rapid drug release in response to the tumor tissue pH value (Scheme 1).
2. Experimental Section 2.1. Materials Polyethylene glycol (PEG, M n ¼ 2000), 4-Nitrophenyl chloroformate, Polyethylenimine (PEI, branched, M w 25000, 25% NH2, 50% NH, 25% N,) and 1,2-cis-cyclohexanedicarboxylic anhydride were purchased from Sigma–Aldrich. Acetonitrile, triethylamine, and ethyl ether were refuxed before using. Doxorubicin hydrochloride (DOX HCl, 99%) was purchased from Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. DOX HCl was neutralized by triethylamine to remove the hydrochloride and make hydrophobic DOX in DMSO. Other reagents were used as received.
2.2. Synthesis of PEG-Nitrophenyl Carbonate (PEG-NO2) The activation of PEG-OH with 4-nitrophenyl chloroformate to generate PEG-NO2, was synthesized according the literature.[35] Briefly, PEG (10 g, 5 mmol) and triethylamine (1.4 mL, 10 mmol) were first added in to a three-necked flask with100 mL acetonitrile. Then, 4-nitrophenyl chloroformate (2 g, 10 mmol) dissolved in 30 mL actonitrile was added into the flask by a drop funnel. The mixture was reacted under 5 8C for 24 h and further freezed under
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Scheme 1. Schematic illustration of the pH-responsive charge conversional and drug release PEG-PEI/amide micelle for effectively drug delivery.
20 8C for 12 h to precipitate triethylamine salt. At last, the filtrate was precipitated in ethy ether to obtain the final product PEG-NO2.
2.3. Synthesis of PEG Grafted Polyethylenimine (PEG-g-PEI) Copolymer 1.6 g PEI was dissolved in 30 mL deionized water and added into a 100 mL flask. Then, 0.42 g PEG-NO2 was added into the solution and reacted with PEI under 30 8C for 48 h. After the reaction, the mixture was transfered into a dialysis bag [molecular-weight cutoff (MWCO) ¼ 3500] and dialyzed against distilled water for 3 d to remove p-nitrophenol and unreacted PEG-NO2. The distlled water was replaced every 6 h. The resulting product, PEG-g-PEI was obtained by freeze drying for 3 days.
2.4. Synthesis of PEG-g-PEI/Amide Amphiphilic Copolymers 1.4 g PEG-g-PEI was dissolved in 15 mL of DMSO in a 50 mL flask with a magnetic stirring bar. 1,2-cis-Cyclohexanedicarboxylic anhydride (3.2 g, 20 mmol) was added. The reaction was kept at room temperature with the protection of nitrogen for 72 h. The mixture was then precipitated in diethyl ether. The solid was isolated and purified by reprecipitation twice. The resulting product, PEG-g-PEI/ amide was dried under high vacuum at 40 8C for 12 h.
2.5. Micelle Preparation and Critical Micelle Concentration (CMC) Nano-sized micelles were prepared via dialysis method. Briefly, 20 mg PEG-g-PEI/amide amphiphilic copolymers were first dis-
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solved in 2 mL DMSO. The mixture was stirred for 2 h at room temperature to ensure complete polymer solubilization. Doubly distilled water from a Milli-Q was then added to the stirring copolymer solution with a microliter syringe at a rate of one drop every 5 s. The total volume of added water was about 1 mL. The resulting aggregate solution was then placed into a dialysis bag (MWCO ¼ 3500) and dialyzed against distilled water for 3 d to remove the organic solvents. The distilled water was replaced every 6 h. The CMC of PEG-g-PEI/amide micelle was determined using pyrene as a fluorescence probe. The concentration of copolymer was varied from 1.0 106 to 1 mg mL1 and the concentration of pyrene was fixed at 0.6 mM. The fluorescence spectra were recorded using Perkin-Elmer LS-55 fluorescence spectrometer with the emission wavelength of 394 nm. The excitation fluorescence at 300–360 nm was monitored. The CMC was estimated as the cross point when extrapolating the intensity ratio I339/I334 at low and high concentration regions.
2.6. Anti-Cancer Drug Loading and Release In Vitro Hydrophobic DOX was loaded into the micelle core during the micelle formation. Briefly, 20 mg PEG-g-PEI/amide amphiphilic copolymers were dissolved in 4 mL DMSO containing 4 mg DOX. Then, 1 mL PBS solution was added dropwise to the mixture, followed by dialysis against PBS for 48 h at room temperature (MWCO ¼ 3500) to remove the unloaded drugs and DMSO. The whole procedure was performed in the dark. The amount of DOX was determined using a UV–vis Shimadzu UV-2502 spectrometer at 485 nm. For determination of drug loading content, lyophilized drug-loaded micelles were dissolved in the mixture of DMSO and analyzed with UV spectroscopy, wherein the calibration curve was obtained with DOX in DMSO solutions with different DOX concentrations (Figure S1, Supporting Information). Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: DLCðwt%Þ ¼ ðweight of loaded drug=weight of polymerÞ 100% DLE ð%Þ ¼ ðweight of loaded drug=weight of drug in feedÞ100% The release profiles of 1 mL free DOX HCl solution or DOX from 1 mL drug-loaded micelles with the same drug concentration were studied using a dialysis tube (MWCO ¼ 3500) at 37 in pH 7.4 or pH 6.0 PBS. Briefly, 1 mL free DOX or DOX-loaded micelles was added into a dialysis tube. Then, the dialysis tube was put into a centrifuge tube with 20 mL PBS (pH 7.4 or 6.0). At predetermined time intervals, 2 mL aliquots of the aqueous solution were withdrawn from the release media and another 2 mL fresh PBS was added into
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the release media. The amount of DOX released was monitered using a UV spectrophotometer at 485 nm.
both maintained at 5.0 nm and spectra were accumulated with a scan speed of 200 nm min1.
2.7. Cytotoxicity Assay
3. Results and Discussion
The cytotoxic effects of PEG-g-PEI/amide micelles were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Briefly, HepG2 cells were plated at 1.0 104 cells per well in a 96-well plate in Dubecco’s modified Eagle medium (DMEM) culture medium supplemented with 10% fetal bovine serum, antibiotics penicillin (100 IU mL1), and streptomycin (100 mg mL1) at 37 8C in a humidified atmosphere containing 5% CO2. After 24 h culture, PEG-g-PEI/amide micelles were added to the 96-well plate with different concentrations, and incubated for 24 h. PBS was chosen as the control. The, 10 mL of a stock solution containing 10 mg mL1 of MTT in PBS was added and incubated for another 4 h. The medium was aspirated, the MTT-formazan generated by live cells was dissolved in DMSO, and the absorbance at a wavelength of 490 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 490 nm with control wells. The cell viabilities of HepG2 cells incubated with blank micelles, DOXloaded micelles, and free DOX were also assessed with MTT assay against HepG2 cells.
2.8. Intracellular Release of DOX The cellular uptake and intracellular release behaviors of DOXloaded PEG-g-PEI/amide micelles was observed by fluorescence microscopy. HepG2 cells (1.0 104 cells per well) were seeded in a 96-well tissue culture plate. After 24 h culture, DOX-loaded micelles or DOX in free form dissolved in complete DMEM with equal amount of DOX (10 mg mL1) were added to distinct wells and the cells were incubated for 4 h at 37 8C. After washing six times with PBS, cells were observed by fluorescence microscopy (Olympus IX71).
2.9. Characterizations Fourier transform infrared (FTIR) spectroscopy was recorded on a Nicolet Nexus 670 FTIR spectrophotometer at room temperature in the range of 4000 and 500 cm1. The solid samples were thoroughly mixed collected over 24 scans with a spectral resolution of 4 cm1. 1 H NMR spectra of the polymers were recorded in DMSO-d6 or D2O using a Bruker DMX500 spectrometer and scanned in the range of 0–15 ppm, the concentration of the polymer was 20 mg mL1. UV–vis spectra of sc-DNQ with different concentrations and the polymers after irradiation for different time were carried out with a UV–vis Shimadzu UV-2505 spectrometer. Spectra were collected within a range of 300–600 nm. The average diameter and size distributions of the micelles were determined using a Zetasizer analyzing system. TEM samples were prepared by drying a drop of a dilute aqueous solution of micelles onto a carbon-coated copper grid. TEM measurements were performed on a JEM-1200EX TEM operating at 80 kV in bright field mode and a staining agent ere used in TEM images. Fluorescence measurements were taken at an excitation wavelength of 420 nm and the emission slit widths were
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3.1. Synthesis of PEG-g-PEI/Amide Amphiphilic Copolymers The detailed synthesis procedure of PEG-g-PEI/amphiphilic copolymer is shown in Scheme 2. First, hydrophilic and biocompatible PEG was activated to generate PEG-NO2 which can react with the amino groups of the PEI. It can be seen from Figure 1a that the signals located at d 3.1 ppm and d 3.6 ppm belonged to the protons of methyl and methylene groups of PEG, respectively. While d 7.4 ppm and d 8.3 ppm were attributed to the protons of nitrobenzene. The ratio of the peak areas at d 7.4 to d 3.1 ppm was 2:3, indicating the PEG-NO2 was synthesized successfully. FTIR spectroscopy provided further evidence for the successful synthesis of PEG-NO2. In the IR spectra, the bands at 1110 and 2888 cm1 were corresponding to the antisymmetric stretching of CH2 and the stretching of C—O—C moieties on the PEG chain, respectively. The bands at 843 and 963 cm1 were due to the bending of C—H on benzene. While bands at 1768, 1344, and 1470 cm1 are due to the stretching of CO and the antisymmetric stretching NO2 moieties. PEG-g-PEI copolymers were prepared through in the presence of PEG-NO2, followed by reacting with amino groups on PEI chains. During the process of reaction, the nitrophenol droped from PEG and amide bonds formed between PEG and PEI. Therefore, PEG can be grafted on to the PEI chain. 1H NMR spectroscopy was employed to prove the successful synthesis of the copolymer, and the 1H NMR spectrum of PEG-g-PEI copolymer is shown in Figure 2a. The peaks at d3.5 and d2.3–2.8 ppm were assigned to the protons of methylene of PEG and PEI, respectively. Moreover, the characterisc peaks of nitrobenzene can not be oberserve at 7.4 and 8.3 ppm, indicating the successfull synthesis of PEG-g-PEI copolymer. The grafting ratio of PEG could be concluded from the integral ratio of the peaks at d3.5 ppm and d2.3–2.8 ppm, and the degree of the amino substitution was about 1.6% (approximately 9 PEG chains were grafed onto the PEI). After the conjugation, the biocomptibility and hydrophilicity will be significantly promoted due to the PEG’s speciality. PEG-g-PEI/amide amphiphilic copolymer was synthesized by modifing the PEI block with hydrophobic cis-1,2-cyclohexanedicarboxylic anhydride to convert the primary and secondary amines into their amides. Figure 2a showed the 1H NMR result of PEG-g-PEI/amide in DMSO-d6. Signals between d1.0 and d2.0 ppm were belong to the protons on the cyclohexane, while the peak at d12 ppm was attributed to
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Scheme 2. Detailed synthetic route of PEG-g-PEI/amide amphiphilic copolymers.
the protons of carboxyl transformed from the hydrolysis of the anhydride. The 1H NMR evidence proved anhydride was conjugated on to the PEI block, and the amidization degree of the PEI was about 35%. That means the PEI block with 35% of its primary and secondary amines converted into their amides. 3.2. Self-Assembly of PEG-g-PEI/Amide Amphiphilic Copolymer Through dialysis against PBS (pH 7.4), polymeric nano-sized micelles can be formed by self-assembling of PEG-g-PEI/
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amide copolymers with amide as hydrophobic cores and PEG-g-PEI as hydrophilic shells. In fact, the self-assembly of amphiphilic PEG-g-PEI/amide gave spherical micelles with a diameter of around 100 nm (Figure 3a). The DLS result in Figure 3b showed the size of the self-assembled micelles was 102 nm with PDI of 0.105. The normalized autocorrelation curves also indicated the spherical structure of this well-formed micelle (Figure S2, Supporting Information). Moreover, the zeta potential of this micelle in pH 7.4 PBS was about 15.5 mV. That is because the cis-1,2-cyclohexanedicarboxylic anhydride exposed one carboxyl groups per reacted amine group, and the anionic charge
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Figure 1. a) 1H NMR spectra of PEG-NO2 in D2O and b) FT-IR spectra of PEG-NO2.
Figure 2.
1
H NMR spectra of PEG-g-PEI copolymers in D2O a) and PEG-g-PEI/amide amphiphilic copolymer in DMSO-d6 b).
desity could be reversibly increased and resulted a negative micelle surface. Detailed morphology analysis was futher investigated by transmission electron microscopy (TEM) and polymeric naoparticles can be clearly seen from the image (Figure 3a). The average diameter roughly calculated by the TEM image is about 90 nm. This value is smaller than the size determined by DLS due to the dry conditon. Thus, the TEM result is consistent with the formation of the DLS result, provided the formation of the well-defined spherical structure. As drug delivery vehicle, polymeric carriers must keep their structure in the blood circulation with very low polymer concentration. Therefore, The CMC value is a critical property of micelles for drug delivery applications. The CMC value can be determined by fluorescence spectrophotometry measurements. Pyrene was chosed as a hydrophobic probe beacuse the redshift of the pyrene parttioning changed its surroundings, from aqueous media into a hydrophobic structure as micellization occurred.
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With increasing concentration, the fluorescence intensity increased and a red shift from 334 to 339 nm was observed. The result indicated the self-assembly of PEG-g-PEI/amide to a micelle structure. The intensity ratio I339/I334 versus logarithm of concentration (Log C) was plotted in Figure S3, Supporting Information. The CMC was obtained from the intersection of the tangent to the curve at the point of inflection with the horizontal tanget through the points at low polymer concentration. The value of the CMC is about 0.026 mg/mL which was lower than that of common surfactant, which would be conducive to further applications such as drug delivery.[36] 3.3. The pH-Sensitive Hydrolysis and Charge Conversion of PEG-g-PEI/Amide Micelle Amides with neighboring carboxylic acid groups usually exhibit a pH-dependent hydrolysis behavior. Recently, the hydrolysis of amide bonds formed between primary or
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Figure 3. The size distribution and TEM image of PEG-g-PEI/amide micelles.
secondary amines and cis-1,2-cyclo-hexanedicarboxylic anhydride have been tested at different pH conditions.[31] The result showed the amide of the secondary amine hydrolyzed significantly faster at pH 6.0 than that at pH 7.4. While the amide of the primary amine hydrolyzed more slowly at pH 6.0 than that of the secondary amine amide, and almost did not hydrolyze at pH 7.4. In this study, this type of amides was used to preserve the primary and secondary amines of PEGylated PEI. At neutral pH, the amides are hydrophobic to be as micelle core and negatively charged because of the b-carboxylic acid groups. While at low pH, the amides are hydrolyzed to regenerate the amine groups to be positively charged and disassemble the micelle. The hydrolysis of PEG-g-PEI/amide micelle was investigated in pH 7.4 and 6.0 PBS at 37 8C, respectively. pH 7.4 was used to mimic the neutral blood pH, and pH 6.0 was similar to tumor tissue or early endosome pH enviroment. It is expected that the amides are stable at the normal physiological pH value of 7.4, but degrade at the tumor pH
value of 6.0 to expose amines, with charge conversion from negative to positive and simultaneous micelle disassembly. The charge-conversion behavior of the PEG-g-PEI/amide micelle was monitored from the changes in the zeta potential, as illustrated in Figure 4a. The zeta potential of micelles at pH 7.4 and 6.0 were both increased. Beacause of the slow degradation of amide moieties at pH 7.4, the micelles still did not convert to a obvious positive zeta potential even after 24 h. However, the zeta potiential at pH 6.0 increased gradually from negative to positive. After incubation for 4 h at pH 6.0, the zeta potential dramatically reached 0 mV, indicating the charge conversion due to the fast hydrolysis. At 6 h under pH 6.0, the zeta potential value of the micelles was about þ4.0 mV, which proved the sucessful charge-conversion driven by the acidic pH. The size changes of micelles in response to amide hydrolysis at different pH conditions was followed by DLS measurements. As shown in Figure 4b, placement of micelles into pH 6.0 buffer resulted in rapid and remarkable swelling of
Figure 4. The size and zeta potential of the PEG-g-PEI/amide micelles at 37 8C as a function of time at different pH values.
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micelles. There was an immediate increase in its size from 100 nm to ca. 450 nm in 24 h. The reason for the size swelling is due to the degradation of amides and the reduction of hydrophobic interaction in micelle core. It is worth noting that from 6 to 24 h at pH 6.0, the zeta potential value of micelles did not increase a lot comparing to the dramatic size increase. This probably because the fast destruction of the particle structure, and the zeta-potential value cannot be accurately reflected by the Zetasizer instrument. In contrast, only a little increase of micelle size was oberved within 6 h at pH 7.4 and then the micelle maintained a diameter of around 200 nm even after 24 h. The charge conversion and pH-sensitive are important merits and give such PEGylated micelle great potential in drug delivery application: With a biocompatible PEG and negative surface, drug-loaded micelle can be long-circulating with blood and resulted in EPR effect; then the acidic pH environment of tumor tissue can simultaneously lead to surface charge conversion, effectively cell-uptaken and pHtriggered drug release; Moreover, drug-loaded micelles with positive charges in early endosome is helpful for escaping and further delivering drugs into cell nuclei. 3.4. Drug Loading and pH-Sensitive Release of Hydrophobic DOX Dox is one of the most potent anti-cancer drugs used widely in the treatment of different types of solid malignant tumors. DOX was loaded into the micelle core during the micelle formation and unencapsulated DOX was removed by dialysis. TEM was used to observe the dry DOX-loaded micelles stained with uranyl acetate, shown in Figure 5a. It was apparent that the micelles still kept their micelluar structure after drug loading. Remarkably, the size of DOXloaded micelles was much smaller than 90 nm of blank micelles observed in Figure 3a. To compare the size changes
Figure 6. Percentage of cumulative release DOX from PEG-g-PEI/ amide micelle at different pH.
after drug loading, the size distribution of DOX-loaded micelles were further measured by artificially analyzing 200 random micelles from the TEM images (displayed in Figure 5b). The result revealed the average diameters of drug-loaded micelles were 25.0 2.1 nm, indicating the significant shrinkage of micelles after drug loading. The size shrinkage was proablely caused by the enhanced hydrophobic interaction between DOX and amides. After drug loading, the micelle core became more hydrophobic and compact. Such relatively small size (25 nm) is ideal for carriers to evade scavenging by the mononnuclear phagocyte system (MPS) and attain an appreciable enhanced permeability and retention (EPR) effect in the site of a solid tumor. In this work, the theoretical drug loading content was set at 20%. For determining the actual drug loading
Figure 5. a) TEM image of dried drug-loaded micelles, followed by staining with uranyl acetate, and the scale bar is 50 nm; b) size distribution measured from the microscopy studies, and the result was based on analysis of 200 micelles.
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content (DLC) and drug loading efficiency (DLE), lyophilized DOX-loaded micelles were dissoved in the DMSO solution and analyzed with UV spectroscopy, wherein calibration curve was obtained with DOX in DMSO with different DOX concentrations (Figure S1, Supporting Information). Properties of the DOX-loaded micelles were summarized in Table S1, Supporting Information, and the results showed the micelles had DLC and DLE of 8% and 45.4%. respectively. The release studies were carried out at 37 8C in pH 7.4 and 6.0 PBS. The results revealed
a significantly faster release of DOX from PEG-g-PEI/ amide micelles at mildly acidic pH 6.0 compared to physiological pH 7.4 (Figure 6), in agreement with the hydrolysis results. Within 72 h, approximately 67% DOX was released at pH 6.0. Whereas at pH 7.4, only 20% DOX was released. It was apparent that the drug release appeared pH-dependent. Such a highly pH-triggered release behavior was attributed to the hydrolysis of amide, which could result in disassembly of micelle with triggered release of encapsulated drug molecules.
Figure 7. Fluorescent images of HepG2 cells incubated with free drug (A1–A3) and drug loaded micelles (B1–B3) for 4 h, respectively. The drug concentration is 10 mg mL1. The scale bars represent 50 mm in the fluorescent micrographs. The images from top to down show DOX fluorescence in cells (red), cell nuclei stained by DAPI (blue), and overlays of the two images.
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Moreover, compared with free DOX HCl release at pH 7.4, the DOX-loaded micelles showed a sustained release at pH 7.4 but a close release rate to free drug at pH 6.0. The above results indicated PEG-g-PEI/amide micelles are appropriate vhicles for anti-cancer drug delivery: drugloaded micelles cycle in the bloodstream for a long time with fewer drug leaks and lower side effect, and then fast release dugs to kill cancer cells after passive accumulation into the tumor tissue with acidic pH. 3.5. Cell Uptake and Intracellular Release of DOX The cellular uptake of the DOX-loaded PEG-g-PEI/amide micelles by HepG2 cells were studied using fluorescence microscopy, and free DOX was used as a control. Remarkably, free DOX was mainly accumulated in cell nucleus with pink fluorescence overlapped by red DOX and blue nucleus (Figure 7A). On the contrary, DOX-loaded micelles efficiently delivered and released the DOX into the cytoplasm (Figure 7B). That because free DOX was transported into cells via diffusion through the cell membrane, while DOXloaded nanoparticles were taken up via an endocytosis process. From the in vitro release study, we have known that at the acidic pH, DOX could be released faster from micelles than that at neutral pH. It can be seen that red DOX fluorescence was clearly observed around the blue nucleus of each HepG2 cell after incubation DOX-loaded micelles for 4 h, thus corroborating fast cell-uptaken and pH-triggered drug release of micelles in the acidic endosomal compartment (Figure 7B3). Therefore, such kind of drug-loaded micelle was able to enhance the intracellular release of DOX. It should be noted, however, that after 4 h incubation, DOX fluorescence could also be observed in the cell nucleus of cells treated with DOX-loaded micelles (see Figure S4, Supporting Information). Pink spots shown by the arrows correspond to DOX colocalized in the nuclei. The further intracellular DOX release into nuclei was presumably facilitated by the micelle degradation and fast drug release from cytoplasm to nuclei. The hydrolysis result has revealed that micelles started to perform a charge-conversion behavior after 4 h at pH 6.0. Thus, after the cellular internalization of drug-loaded micelles for 4 h, some of the endocytosed micelles might begin to express positive charges to disrupt the endosome at endosomal pH values and simultaneously enhanced delivery of drugs from tumor cell cytosol to nuclei.
Figure 8. Cytotoxicity of PEG-g-PEI/amide micelles after 24 h incubation in HepG2 cells. Data are presented as the average SD (n ¼ 3).
0.2 mg mL1. The biocompatible PEG shell and negative charge surface was believed to contribute the excellent biocompatibility. The cytotoxicity of free DOX and DOX-loaded micelles with various DOX concentration against HepG2 cells after 48 h incubation were studies using the MTT assay (Figure 9). Notably, cytotoxicity studies demonstrated that DOX-loaded micelles with higher DOX concentration (10 mg mL1) had a similar drug efficacy as the free DOX, with approximately 80% cell death achieved in 48 h. However, for in vivo applications, it is unlikely that such a high concentration of free DOX would be present for such a long treatmemt time. Nevertheless, at lower
3.6. Cytotoxicity of the PEG-g-PEI/Amide Micelles The tumor HepG2 cells were used to test the cytotoxicity of blank PEG-g-PEI/amide micelles. AS shown in Figure 8, the cytotoxicity of the micelles to HepG2 cells was very low, with over 90% cells viable even at highest concentration of
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Figure 9. In vitro inhibition to HepG2 cell proliferation in 48 h by DOX-loaded micelles and DOX in free form at various does. Data are presented as the average SD (n ¼ 3).
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DOX concentration (
Charge-Conversional and pH-Sensitive PEGylated Polymeric Micelles as Efficient Nanocarriers for Drug Deliverya Gong-Yan Liu, Min Li, Cong-Shan Zhu, Qiao Jin, Zong-Cai Zhang,* Jian Ji*
A novel amphiphilic copolymer, poly (ethylene glycol)-graft-polyethyleneimine/amide (PEGg-PEI/amide), is synthesized by grafting PEG and1,2-cis-Cyclohexanedicarboxylic anhydride onto the PEI. PEGylated polymeric micelles can be assembled from the amphiphilic copolymers with well-defined nano-sizes, and anticancer drugs are successfully loaded into micelle core formed by the amide. The amides with neighboring carboxylic acid groups exhibit pH-dependent hydrolysis and can reversibly shield the cationic charge of amine groups on the PEI, giving the micelles a charge-conversion property from negative to positive in acidic tumor tissue environment. Meanwhile, the cleavage of amide bonds at acidic pH also results in the disassembly of the micelle and pH-responsive drug release. These micelles are promising drug delivery systems due to their smart properties: PEGylation, suitable size, charge-conversion, and simultaneous pHsensitive drug release.
1. Introduction Dr. G.-Y. Liu, M. Li, Prof. Z.-C. Zhang Department of Biomass Chemistry and Engineering, National Engineering Laboratory for Clean Technology of Leather Manufacture, Sichuan University, Chengdu 610065, China E-mail: [email protected] C.-S. Zhu, Q. Jin, Prof. J. Ji MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, Hangzhou 310027, China E-mail: [email protected] Supporting Information is available online from theOnline Library or from the author.
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Over the years, polymeric micelles as nano-scale drug delivery systems have shown great potential for delivering high payloads of therapeutic drugs to tumors.[1–6] Drugloaded polymeric micelles could control the time-dependent drug distribution in the body for improved therapeutic benefits and also reduced adverse effects.[7,8] However, for successful cancer treatment, drug carriers must overcome several transport barriers from systemic administration to access target sites, for example, rapid filtration in the kidney and clearance via the reticulo-endothelial system (RES) as well as transport from the bloodstream into target tissues or cells with controlled drug release.[9–13] Therefore, polymeric
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DOI: 10.1002/mabi.201400162
Charge-Conversional and pH-Sensitive PEGylated Polymeric . . . www.mbs-journal.de
micelles are supposed to be designed with smart properties, such as, reduced inherent toxicity, longevity in blood circulation, tissue accumulation and penetration, and stimuli-responsive drug release.[14–18] To address the renal excretion from kidney and capture by the RES, polymeric micelles with several tens nanometer and biocompatible PEG shell have been developed.[19–22] Through the systemic injection into blood circulation, the nano-scale size (10–100 nm) of micelles can effectively avoid rapid renal excretion and also allow micelles to accumulate into the tumor tissues by the enhanced permeability and retention (EPR) effect.[23] While the ‘‘stealth’’ property of PEGylated micelle will minimize the non-specific interactions with biological components including entrapment by RES, leading to prolonged blood circulation, enhanced passive tumor accumulation, and reduced the toxicity of the drug-loaded micelles.[12] After the accumulation at the target tissue with an acidic pH environment (pH 6.0),[24] micelles must furthermore cross the plasma membrane barriers into cancer cells and enable anti-cancer drugs to be released in a controlled manner. It has been reported that nanoparticles with a positive charged surface showed higher affinity for negative charged cell membranes and thus can be readily internalized by cells.[25] However, the positively charged surface may induce nonspecific interactions with serum, causing high cytotoxicity and excessive immune responsive.[26] On the contrary, negatively charged drug carriers have shown potential for protein resistance and exhibited prolonged circulation time for in vivo application.[27] Therefore, much effort has been concentrated on the development of surface charge-conversion methods triggered by the pH biosignals for drug delivery.[28–33] Recently, Kataoka and co-workers have proposed novel strategy for site-specific protein and gene release from smart polyion complex (PIC) micelles with a charge-conversion property from negative to positive in late endosomal/lysosomal acidic environments.[28–30] Shen et al. have also demonstrated a negative-to-positive charge-reversal technique for preparing polymeric micelles and conjugates for nuclear drug delivery, which could effectively enhance the cellular uptake of the nanoparticles and thereafter direct the carriers to localize in the nucleus.[31,32] Latter, Wang’s group has developed a tumor pH-responsive charge-conversional nanogel for promoted tumoral-cell uptake and Doxorubicin (DOX) delivery.[33] Despite the advantages of charge-conversion polymeric micelles, the simultaneously site-specific release of payload from the carrier is still one of the major obstacles in the development of an ideal drug delivery system. The capabilities of surface charge-conversion and simultaneous drug release in the target site can only be obtained by a smart delivery system that can respond to the change of biosignals, such as pH. In this study, we designed a
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PEGylated polymeric micelle with pH-sensitivity and simultaneous charge-conversion property for efficient anti-cancer drug delivery. PEI with high amine density was chose as the polycations due to its ‘‘proton-sponge’’ effect for tissue penetration.[34] Then, PEGylation was carried out by grafting PEG onto the PEI chain to increase the biocompatibility and reduce the toxicity of PEI. The PEG-gPEI copolymer was furthermore modified by reacting with 1,2-cis-Cyclohexanedicarboxylic anhydride to form PEG-gPEI/amide amphiphilic copolymers, which can self-assembled into polymeric micelles in aqueous solution. The amide bond obtained between the amine groups and anhydride had anionic charges with neighboring carboxylic acid groups, shielding the cationic charge of amine groups on the PEI. The resulting amide was stable at neutral pH, but would be cleaved at tumor pH[31] to revert back the initial amine groups and disassemble the formed micelles with the fast payload release. Therefore, modification with 1,2-cisCyclohexanedicarboxylic anhydride resulted in polymeric micelles having charge-conversional and pH-sensitive properties in tumor tissue acidic environments. After drug loading, such kind of PEGylated polymeric micelles with several tens of nanometer and negative charge surface would possess longevity in blood circulation and achieve successful tissue accumulation via EPR effect. In the following stage, the micelles could be activated to be positively charged for enhanced cellular internalization and rapid drug release in response to the tumor tissue pH value (Scheme 1).
2. Experimental Section 2.1. Materials Polyethylene glycol (PEG, M n ¼ 2000), 4-Nitrophenyl chloroformate, Polyethylenimine (PEI, branched, M w 25000, 25% NH2, 50% NH, 25% N,) and 1,2-cis-cyclohexanedicarboxylic anhydride were purchased from Sigma–Aldrich. Acetonitrile, triethylamine, and ethyl ether were refuxed before using. Doxorubicin hydrochloride (DOX HCl, 99%) was purchased from Beijing Zhongshuo Pharmaceutical Technology Development Co., Ltd. DOX HCl was neutralized by triethylamine to remove the hydrochloride and make hydrophobic DOX in DMSO. Other reagents were used as received.
2.2. Synthesis of PEG-Nitrophenyl Carbonate (PEG-NO2) The activation of PEG-OH with 4-nitrophenyl chloroformate to generate PEG-NO2, was synthesized according the literature.[35] Briefly, PEG (10 g, 5 mmol) and triethylamine (1.4 mL, 10 mmol) were first added in to a three-necked flask with100 mL acetonitrile. Then, 4-nitrophenyl chloroformate (2 g, 10 mmol) dissolved in 30 mL actonitrile was added into the flask by a drop funnel. The mixture was reacted under 5 8C for 24 h and further freezed under
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Scheme 1. Schematic illustration of the pH-responsive charge conversional and drug release PEG-PEI/amide micelle for effectively drug delivery.
20 8C for 12 h to precipitate triethylamine salt. At last, the filtrate was precipitated in ethy ether to obtain the final product PEG-NO2.
2.3. Synthesis of PEG Grafted Polyethylenimine (PEG-g-PEI) Copolymer 1.6 g PEI was dissolved in 30 mL deionized water and added into a 100 mL flask. Then, 0.42 g PEG-NO2 was added into the solution and reacted with PEI under 30 8C for 48 h. After the reaction, the mixture was transfered into a dialysis bag [molecular-weight cutoff (MWCO) ¼ 3500] and dialyzed against distilled water for 3 d to remove p-nitrophenol and unreacted PEG-NO2. The distlled water was replaced every 6 h. The resulting product, PEG-g-PEI was obtained by freeze drying for 3 days.
2.4. Synthesis of PEG-g-PEI/Amide Amphiphilic Copolymers 1.4 g PEG-g-PEI was dissolved in 15 mL of DMSO in a 50 mL flask with a magnetic stirring bar. 1,2-cis-Cyclohexanedicarboxylic anhydride (3.2 g, 20 mmol) was added. The reaction was kept at room temperature with the protection of nitrogen for 72 h. The mixture was then precipitated in diethyl ether. The solid was isolated and purified by reprecipitation twice. The resulting product, PEG-g-PEI/ amide was dried under high vacuum at 40 8C for 12 h.
2.5. Micelle Preparation and Critical Micelle Concentration (CMC) Nano-sized micelles were prepared via dialysis method. Briefly, 20 mg PEG-g-PEI/amide amphiphilic copolymers were first dis-
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solved in 2 mL DMSO. The mixture was stirred for 2 h at room temperature to ensure complete polymer solubilization. Doubly distilled water from a Milli-Q was then added to the stirring copolymer solution with a microliter syringe at a rate of one drop every 5 s. The total volume of added water was about 1 mL. The resulting aggregate solution was then placed into a dialysis bag (MWCO ¼ 3500) and dialyzed against distilled water for 3 d to remove the organic solvents. The distilled water was replaced every 6 h. The CMC of PEG-g-PEI/amide micelle was determined using pyrene as a fluorescence probe. The concentration of copolymer was varied from 1.0 106 to 1 mg mL1 and the concentration of pyrene was fixed at 0.6 mM. The fluorescence spectra were recorded using Perkin-Elmer LS-55 fluorescence spectrometer with the emission wavelength of 394 nm. The excitation fluorescence at 300–360 nm was monitored. The CMC was estimated as the cross point when extrapolating the intensity ratio I339/I334 at low and high concentration regions.
2.6. Anti-Cancer Drug Loading and Release In Vitro Hydrophobic DOX was loaded into the micelle core during the micelle formation. Briefly, 20 mg PEG-g-PEI/amide amphiphilic copolymers were dissolved in 4 mL DMSO containing 4 mg DOX. Then, 1 mL PBS solution was added dropwise to the mixture, followed by dialysis against PBS for 48 h at room temperature (MWCO ¼ 3500) to remove the unloaded drugs and DMSO. The whole procedure was performed in the dark. The amount of DOX was determined using a UV–vis Shimadzu UV-2502 spectrometer at 485 nm. For determination of drug loading content, lyophilized drug-loaded micelles were dissolved in the mixture of DMSO and analyzed with UV spectroscopy, wherein the calibration curve was obtained with DOX in DMSO solutions with different DOX concentrations (Figure S1, Supporting Information). Drug loading content (DLC) and drug loading efficiency (DLE) were calculated according to the following formula: DLCðwt%Þ ¼ ðweight of loaded drug=weight of polymerÞ 100% DLE ð%Þ ¼ ðweight of loaded drug=weight of drug in feedÞ100% The release profiles of 1 mL free DOX HCl solution or DOX from 1 mL drug-loaded micelles with the same drug concentration were studied using a dialysis tube (MWCO ¼ 3500) at 37 in pH 7.4 or pH 6.0 PBS. Briefly, 1 mL free DOX or DOX-loaded micelles was added into a dialysis tube. Then, the dialysis tube was put into a centrifuge tube with 20 mL PBS (pH 7.4 or 6.0). At predetermined time intervals, 2 mL aliquots of the aqueous solution were withdrawn from the release media and another 2 mL fresh PBS was added into
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the release media. The amount of DOX released was monitered using a UV spectrophotometer at 485 nm.
both maintained at 5.0 nm and spectra were accumulated with a scan speed of 200 nm min1.
2.7. Cytotoxicity Assay
3. Results and Discussion
The cytotoxic effects of PEG-g-PEI/amide micelles were determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT) assay. Briefly, HepG2 cells were plated at 1.0 104 cells per well in a 96-well plate in Dubecco’s modified Eagle medium (DMEM) culture medium supplemented with 10% fetal bovine serum, antibiotics penicillin (100 IU mL1), and streptomycin (100 mg mL1) at 37 8C in a humidified atmosphere containing 5% CO2. After 24 h culture, PEG-g-PEI/amide micelles were added to the 96-well plate with different concentrations, and incubated for 24 h. PBS was chosen as the control. The, 10 mL of a stock solution containing 10 mg mL1 of MTT in PBS was added and incubated for another 4 h. The medium was aspirated, the MTT-formazan generated by live cells was dissolved in DMSO, and the absorbance at a wavelength of 490 nm of each well was measured using a microplate reader. The relative cell viability (%) was determined by comparing the absorbance at 490 nm with control wells. The cell viabilities of HepG2 cells incubated with blank micelles, DOXloaded micelles, and free DOX were also assessed with MTT assay against HepG2 cells.
2.8. Intracellular Release of DOX The cellular uptake and intracellular release behaviors of DOXloaded PEG-g-PEI/amide micelles was observed by fluorescence microscopy. HepG2 cells (1.0 104 cells per well) were seeded in a 96-well tissue culture plate. After 24 h culture, DOX-loaded micelles or DOX in free form dissolved in complete DMEM with equal amount of DOX (10 mg mL1) were added to distinct wells and the cells were incubated for 4 h at 37 8C. After washing six times with PBS, cells were observed by fluorescence microscopy (Olympus IX71).
2.9. Characterizations Fourier transform infrared (FTIR) spectroscopy was recorded on a Nicolet Nexus 670 FTIR spectrophotometer at room temperature in the range of 4000 and 500 cm1. The solid samples were thoroughly mixed collected over 24 scans with a spectral resolution of 4 cm1. 1 H NMR spectra of the polymers were recorded in DMSO-d6 or D2O using a Bruker DMX500 spectrometer and scanned in the range of 0–15 ppm, the concentration of the polymer was 20 mg mL1. UV–vis spectra of sc-DNQ with different concentrations and the polymers after irradiation for different time were carried out with a UV–vis Shimadzu UV-2505 spectrometer. Spectra were collected within a range of 300–600 nm. The average diameter and size distributions of the micelles were determined using a Zetasizer analyzing system. TEM samples were prepared by drying a drop of a dilute aqueous solution of micelles onto a carbon-coated copper grid. TEM measurements were performed on a JEM-1200EX TEM operating at 80 kV in bright field mode and a staining agent ere used in TEM images. Fluorescence measurements were taken at an excitation wavelength of 420 nm and the emission slit widths were
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3.1. Synthesis of PEG-g-PEI/Amide Amphiphilic Copolymers The detailed synthesis procedure of PEG-g-PEI/amphiphilic copolymer is shown in Scheme 2. First, hydrophilic and biocompatible PEG was activated to generate PEG-NO2 which can react with the amino groups of the PEI. It can be seen from Figure 1a that the signals located at d 3.1 ppm and d 3.6 ppm belonged to the protons of methyl and methylene groups of PEG, respectively. While d 7.4 ppm and d 8.3 ppm were attributed to the protons of nitrobenzene. The ratio of the peak areas at d 7.4 to d 3.1 ppm was 2:3, indicating the PEG-NO2 was synthesized successfully. FTIR spectroscopy provided further evidence for the successful synthesis of PEG-NO2. In the IR spectra, the bands at 1110 and 2888 cm1 were corresponding to the antisymmetric stretching of CH2 and the stretching of C—O—C moieties on the PEG chain, respectively. The bands at 843 and 963 cm1 were due to the bending of C—H on benzene. While bands at 1768, 1344, and 1470 cm1 are due to the stretching of CO and the antisymmetric stretching NO2 moieties. PEG-g-PEI copolymers were prepared through in the presence of PEG-NO2, followed by reacting with amino groups on PEI chains. During the process of reaction, the nitrophenol droped from PEG and amide bonds formed between PEG and PEI. Therefore, PEG can be grafted on to the PEI chain. 1H NMR spectroscopy was employed to prove the successful synthesis of the copolymer, and the 1H NMR spectrum of PEG-g-PEI copolymer is shown in Figure 2a. The peaks at d3.5 and d2.3–2.8 ppm were assigned to the protons of methylene of PEG and PEI, respectively. Moreover, the characterisc peaks of nitrobenzene can not be oberserve at 7.4 and 8.3 ppm, indicating the successfull synthesis of PEG-g-PEI copolymer. The grafting ratio of PEG could be concluded from the integral ratio of the peaks at d3.5 ppm and d2.3–2.8 ppm, and the degree of the amino substitution was about 1.6% (approximately 9 PEG chains were grafed onto the PEI). After the conjugation, the biocomptibility and hydrophilicity will be significantly promoted due to the PEG’s speciality. PEG-g-PEI/amide amphiphilic copolymer was synthesized by modifing the PEI block with hydrophobic cis-1,2-cyclohexanedicarboxylic anhydride to convert the primary and secondary amines into their amides. Figure 2a showed the 1H NMR result of PEG-g-PEI/amide in DMSO-d6. Signals between d1.0 and d2.0 ppm were belong to the protons on the cyclohexane, while the peak at d12 ppm was attributed to
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Scheme 2. Detailed synthetic route of PEG-g-PEI/amide amphiphilic copolymers.
the protons of carboxyl transformed from the hydrolysis of the anhydride. The 1H NMR evidence proved anhydride was conjugated on to the PEI block, and the amidization degree of the PEI was about 35%. That means the PEI block with 35% of its primary and secondary amines converted into their amides. 3.2. Self-Assembly of PEG-g-PEI/Amide Amphiphilic Copolymer Through dialysis against PBS (pH 7.4), polymeric nano-sized micelles can be formed by self-assembling of PEG-g-PEI/
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amide copolymers with amide as hydrophobic cores and PEG-g-PEI as hydrophilic shells. In fact, the self-assembly of amphiphilic PEG-g-PEI/amide gave spherical micelles with a diameter of around 100 nm (Figure 3a). The DLS result in Figure 3b showed the size of the self-assembled micelles was 102 nm with PDI of 0.105. The normalized autocorrelation curves also indicated the spherical structure of this well-formed micelle (Figure S2, Supporting Information). Moreover, the zeta potential of this micelle in pH 7.4 PBS was about 15.5 mV. That is because the cis-1,2-cyclohexanedicarboxylic anhydride exposed one carboxyl groups per reacted amine group, and the anionic charge
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Figure 1. a) 1H NMR spectra of PEG-NO2 in D2O and b) FT-IR spectra of PEG-NO2.
Figure 2.
1
H NMR spectra of PEG-g-PEI copolymers in D2O a) and PEG-g-PEI/amide amphiphilic copolymer in DMSO-d6 b).
desity could be reversibly increased and resulted a negative micelle surface. Detailed morphology analysis was futher investigated by transmission electron microscopy (TEM) and polymeric naoparticles can be clearly seen from the image (Figure 3a). The average diameter roughly calculated by the TEM image is about 90 nm. This value is smaller than the size determined by DLS due to the dry conditon. Thus, the TEM result is consistent with the formation of the DLS result, provided the formation of the well-defined spherical structure. As drug delivery vehicle, polymeric carriers must keep their structure in the blood circulation with very low polymer concentration. Therefore, The CMC value is a critical property of micelles for drug delivery applications. The CMC value can be determined by fluorescence spectrophotometry measurements. Pyrene was chosed as a hydrophobic probe beacuse the redshift of the pyrene parttioning changed its surroundings, from aqueous media into a hydrophobic structure as micellization occurred.
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With increasing concentration, the fluorescence intensity increased and a red shift from 334 to 339 nm was observed. The result indicated the self-assembly of PEG-g-PEI/amide to a micelle structure. The intensity ratio I339/I334 versus logarithm of concentration (Log C) was plotted in Figure S3, Supporting Information. The CMC was obtained from the intersection of the tangent to the curve at the point of inflection with the horizontal tanget through the points at low polymer concentration. The value of the CMC is about 0.026 mg/mL which was lower than that of common surfactant, which would be conducive to further applications such as drug delivery.[36] 3.3. The pH-Sensitive Hydrolysis and Charge Conversion of PEG-g-PEI/Amide Micelle Amides with neighboring carboxylic acid groups usually exhibit a pH-dependent hydrolysis behavior. Recently, the hydrolysis of amide bonds formed between primary or
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Figure 3. The size distribution and TEM image of PEG-g-PEI/amide micelles.
secondary amines and cis-1,2-cyclo-hexanedicarboxylic anhydride have been tested at different pH conditions.[31] The result showed the amide of the secondary amine hydrolyzed significantly faster at pH 6.0 than that at pH 7.4. While the amide of the primary amine hydrolyzed more slowly at pH 6.0 than that of the secondary amine amide, and almost did not hydrolyze at pH 7.4. In this study, this type of amides was used to preserve the primary and secondary amines of PEGylated PEI. At neutral pH, the amides are hydrophobic to be as micelle core and negatively charged because of the b-carboxylic acid groups. While at low pH, the amides are hydrolyzed to regenerate the amine groups to be positively charged and disassemble the micelle. The hydrolysis of PEG-g-PEI/amide micelle was investigated in pH 7.4 and 6.0 PBS at 37 8C, respectively. pH 7.4 was used to mimic the neutral blood pH, and pH 6.0 was similar to tumor tissue or early endosome pH enviroment. It is expected that the amides are stable at the normal physiological pH value of 7.4, but degrade at the tumor pH
value of 6.0 to expose amines, with charge conversion from negative to positive and simultaneous micelle disassembly. The charge-conversion behavior of the PEG-g-PEI/amide micelle was monitored from the changes in the zeta potential, as illustrated in Figure 4a. The zeta potential of micelles at pH 7.4 and 6.0 were both increased. Beacause of the slow degradation of amide moieties at pH 7.4, the micelles still did not convert to a obvious positive zeta potential even after 24 h. However, the zeta potiential at pH 6.0 increased gradually from negative to positive. After incubation for 4 h at pH 6.0, the zeta potential dramatically reached 0 mV, indicating the charge conversion due to the fast hydrolysis. At 6 h under pH 6.0, the zeta potential value of the micelles was about þ4.0 mV, which proved the sucessful charge-conversion driven by the acidic pH. The size changes of micelles in response to amide hydrolysis at different pH conditions was followed by DLS measurements. As shown in Figure 4b, placement of micelles into pH 6.0 buffer resulted in rapid and remarkable swelling of
Figure 4. The size and zeta potential of the PEG-g-PEI/amide micelles at 37 8C as a function of time at different pH values.
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micelles. There was an immediate increase in its size from 100 nm to ca. 450 nm in 24 h. The reason for the size swelling is due to the degradation of amides and the reduction of hydrophobic interaction in micelle core. It is worth noting that from 6 to 24 h at pH 6.0, the zeta potential value of micelles did not increase a lot comparing to the dramatic size increase. This probably because the fast destruction of the particle structure, and the zeta-potential value cannot be accurately reflected by the Zetasizer instrument. In contrast, only a little increase of micelle size was oberved within 6 h at pH 7.4 and then the micelle maintained a diameter of around 200 nm even after 24 h. The charge conversion and pH-sensitive are important merits and give such PEGylated micelle great potential in drug delivery application: With a biocompatible PEG and negative surface, drug-loaded micelle can be long-circulating with blood and resulted in EPR effect; then the acidic pH environment of tumor tissue can simultaneously lead to surface charge conversion, effectively cell-uptaken and pHtriggered drug release; Moreover, drug-loaded micelles with positive charges in early endosome is helpful for escaping and further delivering drugs into cell nuclei. 3.4. Drug Loading and pH-Sensitive Release of Hydrophobic DOX Dox is one of the most potent anti-cancer drugs used widely in the treatment of different types of solid malignant tumors. DOX was loaded into the micelle core during the micelle formation and unencapsulated DOX was removed by dialysis. TEM was used to observe the dry DOX-loaded micelles stained with uranyl acetate, shown in Figure 5a. It was apparent that the micelles still kept their micelluar structure after drug loading. Remarkably, the size of DOXloaded micelles was much smaller than 90 nm of blank micelles observed in Figure 3a. To compare the size changes
Figure 6. Percentage of cumulative release DOX from PEG-g-PEI/ amide micelle at different pH.
after drug loading, the size distribution of DOX-loaded micelles were further measured by artificially analyzing 200 random micelles from the TEM images (displayed in Figure 5b). The result revealed the average diameters of drug-loaded micelles were 25.0 2.1 nm, indicating the significant shrinkage of micelles after drug loading. The size shrinkage was proablely caused by the enhanced hydrophobic interaction between DOX and amides. After drug loading, the micelle core became more hydrophobic and compact. Such relatively small size (25 nm) is ideal for carriers to evade scavenging by the mononnuclear phagocyte system (MPS) and attain an appreciable enhanced permeability and retention (EPR) effect in the site of a solid tumor. In this work, the theoretical drug loading content was set at 20%. For determining the actual drug loading
Figure 5. a) TEM image of dried drug-loaded micelles, followed by staining with uranyl acetate, and the scale bar is 50 nm; b) size distribution measured from the microscopy studies, and the result was based on analysis of 200 micelles.
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content (DLC) and drug loading efficiency (DLE), lyophilized DOX-loaded micelles were dissoved in the DMSO solution and analyzed with UV spectroscopy, wherein calibration curve was obtained with DOX in DMSO with different DOX concentrations (Figure S1, Supporting Information). Properties of the DOX-loaded micelles were summarized in Table S1, Supporting Information, and the results showed the micelles had DLC and DLE of 8% and 45.4%. respectively. The release studies were carried out at 37 8C in pH 7.4 and 6.0 PBS. The results revealed
a significantly faster release of DOX from PEG-g-PEI/ amide micelles at mildly acidic pH 6.0 compared to physiological pH 7.4 (Figure 6), in agreement with the hydrolysis results. Within 72 h, approximately 67% DOX was released at pH 6.0. Whereas at pH 7.4, only 20% DOX was released. It was apparent that the drug release appeared pH-dependent. Such a highly pH-triggered release behavior was attributed to the hydrolysis of amide, which could result in disassembly of micelle with triggered release of encapsulated drug molecules.
Figure 7. Fluorescent images of HepG2 cells incubated with free drug (A1–A3) and drug loaded micelles (B1–B3) for 4 h, respectively. The drug concentration is 10 mg mL1. The scale bars represent 50 mm in the fluorescent micrographs. The images from top to down show DOX fluorescence in cells (red), cell nuclei stained by DAPI (blue), and overlays of the two images.
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Moreover, compared with free DOX HCl release at pH 7.4, the DOX-loaded micelles showed a sustained release at pH 7.4 but a close release rate to free drug at pH 6.0. The above results indicated PEG-g-PEI/amide micelles are appropriate vhicles for anti-cancer drug delivery: drugloaded micelles cycle in the bloodstream for a long time with fewer drug leaks and lower side effect, and then fast release dugs to kill cancer cells after passive accumulation into the tumor tissue with acidic pH. 3.5. Cell Uptake and Intracellular Release of DOX The cellular uptake of the DOX-loaded PEG-g-PEI/amide micelles by HepG2 cells were studied using fluorescence microscopy, and free DOX was used as a control. Remarkably, free DOX was mainly accumulated in cell nucleus with pink fluorescence overlapped by red DOX and blue nucleus (Figure 7A). On the contrary, DOX-loaded micelles efficiently delivered and released the DOX into the cytoplasm (Figure 7B). That because free DOX was transported into cells via diffusion through the cell membrane, while DOXloaded nanoparticles were taken up via an endocytosis process. From the in vitro release study, we have known that at the acidic pH, DOX could be released faster from micelles than that at neutral pH. It can be seen that red DOX fluorescence was clearly observed around the blue nucleus of each HepG2 cell after incubation DOX-loaded micelles for 4 h, thus corroborating fast cell-uptaken and pH-triggered drug release of micelles in the acidic endosomal compartment (Figure 7B3). Therefore, such kind of drug-loaded micelle was able to enhance the intracellular release of DOX. It should be noted, however, that after 4 h incubation, DOX fluorescence could also be observed in the cell nucleus of cells treated with DOX-loaded micelles (see Figure S4, Supporting Information). Pink spots shown by the arrows correspond to DOX colocalized in the nuclei. The further intracellular DOX release into nuclei was presumably facilitated by the micelle degradation and fast drug release from cytoplasm to nuclei. The hydrolysis result has revealed that micelles started to perform a charge-conversion behavior after 4 h at pH 6.0. Thus, after the cellular internalization of drug-loaded micelles for 4 h, some of the endocytosed micelles might begin to express positive charges to disrupt the endosome at endosomal pH values and simultaneously enhanced delivery of drugs from tumor cell cytosol to nuclei.
Figure 8. Cytotoxicity of PEG-g-PEI/amide micelles after 24 h incubation in HepG2 cells. Data are presented as the average SD (n ¼ 3).
0.2 mg mL1. The biocompatible PEG shell and negative charge surface was believed to contribute the excellent biocompatibility. The cytotoxicity of free DOX and DOX-loaded micelles with various DOX concentration against HepG2 cells after 48 h incubation were studies using the MTT assay (Figure 9). Notably, cytotoxicity studies demonstrated that DOX-loaded micelles with higher DOX concentration (10 mg mL1) had a similar drug efficacy as the free DOX, with approximately 80% cell death achieved in 48 h. However, for in vivo applications, it is unlikely that such a high concentration of free DOX would be present for such a long treatmemt time. Nevertheless, at lower
3.6. Cytotoxicity of the PEG-g-PEI/Amide Micelles The tumor HepG2 cells were used to test the cytotoxicity of blank PEG-g-PEI/amide micelles. AS shown in Figure 8, the cytotoxicity of the micelles to HepG2 cells was very low, with over 90% cells viable even at highest concentration of
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Figure 9. In vitro inhibition to HepG2 cell proliferation in 48 h by DOX-loaded micelles and DOX in free form at various does. Data are presented as the average SD (n ¼ 3).
Macromol. Biosci. 2014, 14, 1280–1290 ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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G.-Y. Liu, M. Li, C.-S. Zhu, Q. Jin, Z.-C. Zhang, J. Ji www.mbs-journal.de
DOX concentration (
