Preparation of Multifunctional Drug Carrier for Tumor-Specific Uptake

May 27, 2009 - We demonstrate that multifuctional drug carriers, e.g., polymeric micelles, for tumor-specific uptake and intracellular delivery can be...
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Bioconjugate Chem. 2009, 20, 1163–1170

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Preparation of Multifunctional Drug Carrier for Tumor-Specific Uptake and Enhanced Intracellular Delivery through the Conjugation of Weak Acid Labile Linker Caixia Ding, Jingxia Gu, Xiaozhong Qu,* and Zhenzhong Yang* State Key Laboratory of Polymer Physics and Chemistry, Institute of Chemistry, Chinese Academy of Sciences, Beijing 100190, China. Received December 24, 2008; Revised Manuscript Received April 19, 2009

We demonstrate that multifuctional drug carriers, e.g., polymeric micelles, for tumor-specific uptake and intracellular delivery can be generated from the pH-dependent progressive hydrolysis of a novel benzoic-imine linker in the micelle-forming amphiphilic polymer. The linker, hence the micelle, is stable at physiological pH, partially hydrolyzes at the extracellular pH of the solid tumor, and completely hydrolyzes at the endosomal pH. Meanwhile, the surface property of the micelle converts from neutral to positively charged due to the generation of amino groups from the cleavage of the imine bond at tumor pH. The ionization on the surface facilitates the cellular uptake of the micelles through the electrostatic interaction between the micelle and the cell membrane. Subsequently, at the endosomal pH, with more complete cleavage of the polymer the micellar structure dissociates, and the system becomes very membrane-disruptive, inferring an enhanced intracellular delivery capability via the endosomal pathway.

INTRODUCTION The ultimate goal of controlled drug delivery is to achieve a desired therapeutic concentration at the targeting site while the drug concentrations at other tissues are kept at safe levels (1). An ideal controlled delivery vehicle for antitumor drugs should have long circulation duration, the ability to catch tumor cells, and eventually the ability to efficiently deliver the drug into the cytoplasm (2, 3). Therefore, a key issue is to construct multifunctional drug carriers with the ability to tune their interactions with different biological membranes following the change of environmental conditions during the entire delivery procedure (4). In addition to the conjugation of various targeting antibodies and ligands (5-8), surface charge conversion of a drug carrier in response to the change of external pH around 6.8-7.4 would also help to gain smart antitumor drug delivery systems, since the extracellular environment of solid tumors is slightly acidic (pH ∼6.8) compared with the physiological pH (7.4) (9-12). Recently, a few such multifunctional antitumor drug delivery systems have been reported based on some charge conversion polymers (4), such as poly(L-histidine) (pKb ) 6.5) (9) and poly(sulfonamide) (pKa ∼ 7.0) (10). On the other hand, in nanosized delivery systems a protective layer on the surface, usually a PEG corona, is required to prevent nonspecific interaction and stimulation to the immune system during circulation (13, 14). However, the PEG corona brings a controversial to hamper the intracellular transport (14, 15). In order to enhance the bioavailability of the therapeutic agents, many strategies have focused on the development of intelligent polymeric colloidal systems and liposomes with low-pH triggered membrane disrupting ability (16-19), because nanosized carriers are mainly taken up by cells via endocytosis and the pH values in endosomes (5.0-6.5) are much lower than the physiological pH (20). It is reasonable to insert acid-cleavable linkers in a nanocarrier to induce the destabilization of the carrier * To whom correspondence should be addressed. Tel.: +86-1082619206; fax: +86-10-62559373, E-mail address: [email protected]; [email protected].

in endosome (21-24), hence the disruption of the endosomal membrane to facilitate the release of incorporated drug into cytoplasm (16). So far, acid-labile covalent bonds such as hydrazone (25-28), acetal (16, 21, 22, 29), and orthoester (30, 31), which hydrolyze rapidly in the endosomal compartment (pH ∼5), are extensively used to build the intracellular delivery systems. However, unlike the charge conversion polymers, tumor specificity has rarely been gained from the inclusion of these linkers, since the gap between physiological pH (7.4) and the extracellular pH of the tumor (6.8) is so subtle that the degree of the linker cleavage at tumor pH could not cause a significant property change of the carriers (17). Imine bond hydrolyzes under very weak acidic conditions; however, it is scarcely used to construct pH-triggered delivery vehicles, since it is unstable at physiological pH (32). However, early studies mainly on Schiff base amphiphiles have shown that π-π conjugation can significantly improve the stability of imine bond in water (33, 34). Thus, the investigation of the pH sensitivity of those structures around physiological pH is interesting and helpful for the design of pH-triggered delivery systems, since the hydrolysis of an imine bond generates an amino group, which could affect the charge property of the system. We have previously studied a benzoic-imine linker and found that it hydrolyzes under very slightly acidic conditions whereas it is stable at neutral and basic pH due to the proper π-π conjugation extent (35). Amphiphilic poly(L-lysine) micelles were PEGylated via the benzoic-imine linker and showed a neutral zeta potential at physiological pH (7.4) but were positively charged under weak acidic conditions. In the present work, another advantage of the benzoic-imine bond was that the hydrolysis level is significantly influenced by the solution pH within a very narrow pH interval (7.4-5.0). In a drug nanocarrier, the pH-dependent hydrolysis of the benzoic-imine bond suggests an internal structural evolution of the carrier, e.g., production of more positively charged groups, with the acidity in body sites such as solid tumors (pH ∼6.8) and endosome (pH ∼5.0-6.5), hence the increase of electrostatic interaction between the carrier and biological membranes. This will probably generate multifunctional nature of the carrier for tumor-

10.1021/bc800563g CCC: $40.75  2009 American Chemical Society Published on Web 05/27/2009

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Figure 1. Schematic representation of a multifunctional drug carrier with tumor-targeting capability and intracellular delivery ability originated from the pH-dependent progressive hydrolysis of a chemical bond, e.g., benzoic-imine, in the carrier-forming polymer. The particle has a PEG corona to obtain prolonged circulation in the blood. A suitable particle size would accumulate the carrier in tumor tissue by the “enhanced permeation and retention effect” (EPR). Under the weak acidic condition in a tumor, the particle surface becomes positively charged due to partial hydrolysis of the imine linker, facilitating cellular uptake through adsorptive endocytosis. Subsequently, in the more acidic environment of the endosome, the complete hydrolysis of the imine bond causes the particle to dissociate and destabilize the endosomal membrane to release the therapeutics rapidly into the cytoplasm.

specific uptake and enhanced intracellular delivery even without the inclusion of a biological ligand. The schematic strategy of such a system, i.e., a model polymeric micelle, is shown in Figure 1.

EXPERIMENTAL PROCEDURES Materials. Methoxy poly(ethylene glycol) (mPEG) (MW ) 2k Da), n-octadecane amine, dicyclohexyl carbodiimide (DCC), 4-(dimethylamino) pyridine (DMAP), sodium borohydride (NaBH4), pyrene, Triton X-100, doxorubicin (DOX), chloroform-d (CDCl3), deuterated water (D2O), and phosphate buffered saline (PBS) were all purchased from Sigma-Aldrich (St. Louis, US). p-Formylbenzoic acid was a generous gift from Beijing Prina Chemical Industry Co., Ltd., China. Solvents and other compounds were obtained form Beijing Chemical Reagents Company, China. All reagents were used as received. Synthesis of Amphiphilic Polymers (PEG-b-C18 and PEGC18). Methoxy poly(ethylene glycol) benzaldehyde (PEG-CHO) was synthesized as previously described (35). Typically, pformylbenzoic acid (6 g, 10 equiv), DCC (8.2 g, 10 equiv), and DMAP (1.2 g, 2.5 equiv) were added to a solution of mPEG (8 g, 1 equiv) in dichloromethane (DCM) (150 mL). After stirring for 24 h, the solution was filtered. The filtrate was concentrated, dissolved in isopropanol (80 mL), and cooled at 0 °C for 2 h. The resulting crystals were collected by filtration and washed with isopropanol and diethyl ether with 80% yield. 1H NMR (CDCl3): δ 10.12 (s, 1H), 8.23 (d, 2H), 7.95 (d, 2H), 3.65 (m, CH2 of PEG), 3.32 (s, 3H). n-Octadecane amine (300 mg, 1 equiv) was dissolved in 10 mL of DMSO. To this solution was added a desired amount of the functionalized PEG (2 g, 0.9 equiv) which was dissolved in 50 mL of DMSO. The mixture was stirred and heated to 40 °C for 4 h before the solvent was evaporated by a rotary evaporator. The rough product was washed with diethyl ether and methanol, and dried under reduced pressure at 40 °C to gain a white powder-like PEG-b-C18 in an 80% yield. 1H NMR (CDCl3): δ 8.34 (d, 1H), 8.10 (d, 2H), 7.80 (d, 2H), 3.66 (m,

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CH2 of PEG), 3.40 (s, 3H), 2.06 (m, 2H), 1.63 (m, 2H), 1.28 (m, CH2 of n-octadecane amine), 0.90 (t, 3H). 200 mg of the PEG-b-C18 (1 equiv) was dissolved in 10 mL of ethanol with stirring followed by the addition of 6.5 mg of NaBH4 (2 equiv) to reduce the imine bond to amine. The mixture was stirred overnight, dialyzed against 0.05 M HCl and then water (molecular weight cutoff 2 kDa), and freeze-dried. The resultant white powder was washed with diethyl ether and dried under reduced pressure to gain the non-acid-labile PEG-C18 as a control polymer of the PEG-b-C18 for the property characterizations. The yield was 70%. 1H NMR (CDCl3): δ 8.05 (d, 2H), 7.53 (d, 2H), 4.37 (m, 2H), 3.65 (m, CH2 of PEG), 3.40 (s, 3H), 2.68 (t, 2H), 1.63 (m, 2H), 1.28 (m, CH2 of n-octadecane amine), 0.90 (t, 3H). Characterization. 1H NMR analysis was performed on polymer solutions in CDCl3 or in deuterated water (D2O) at a required pH adjusted by DCl and/or NaOD using a Bruker AMX 600 MHz spectrometer. Infrared spectroscopy (FTIR) of the polymers was recorded using a Bruker Equinox 55 FT-IR spectrometer. Freeze-dried polymer was pressed with KBr under vacuum and scanned from 4000 to 400 cm-1 with a resolution of 2 cm-1. To monitor the hydrolysis of the benzoic imine linkage at acidic pH, PEG-b-C18 was dispersed in water at an acidic pH (e.g., 5.0, adjusted by adding 0.1 M HCl), and then aliquots of the dispersion were dropped on KBr, dried, and recorded. Micelle Formation of the Amphiphilic Polymers. Polymer samples were dispersed in water or PBS buffer with a desired pH by probe sonication using a JY96-II probe sonicator (Zhejiang Xin-Zhi, China) with the output set at 150 W. The solution pH was checked by a Delta 320 pH meter (MettlerToledo Instrument Ltd., Switzerland) after sonication. Dynamic Light Scattering and Zeta Potential Measurement. Particle size measurement was carried out with a Brookhaven Zetaplus analyzer (Brookhaven Instruments Corporation, US), and the zeta potential was measured using a Zetasizer 3000 HS (Malvern Instruments, UK). All samples were passed through a membrane filter (pore size: 450 nm, Millipore) before detection. Fluorescence Spectrophotometry. Aliquots of pyrene stock solution (1.54 × 10-5 M in acetone, 200 µL) were added to 5 mL volumetric flasks, and acetone was evaporated. Five milliliters of the aqueous polymer solution was then added to the volumetric flasks containing the pyrene residue. The solutions were equilibrated for 4 h at 60 °C. Fluorescence spectra of the polymer solutions were then recorded on a fluorescence spectrometer (Cary Eclipse, Varian, US). The emission spectra were recorded from 350 to 500 nm with an excitation wavelength of 340 nm. The first inflection point of the intensity ratio plot of the third band to the first band (I3/I1) against polymer concentration was determined as the critical micelle concentration (cmc). Drug Loading and Release. Doxorubicin hydrochloride (1-5 mg, 1 equiv) was stirred with TEA (3 equiv) in DMSO for overnight before the solvent was evaporated using a rotary evaporator. An aqueous solution of the polymers (PEG-b-C18 or PEG-C18, 5 mL, 1 mg/mL) was then added, and the mixture was sonicated using a bath sonicator for 20 min. The dispersion was transferred into a dialysis tube (molecular weight cutoff 2 kDa) and dialyzed against water for 24 h at pH ) 7.4. The entrapped drug amount was determined by measuring the UV absorbance at 480 nm (UV-1601PC, Shimadzu, Japan). Freeze-dried drug-loaded micelles were dispersed in PBS at different pHs (7.4 and 6.8). Three milliliters of the dispersion was transferred into a dialysis bag (molecular weight cutoff 2 kDa), and the bag was subsequently placed in a 50 mL flask containing 30 mL of PBS adjusted to a required pH. One

Multifunctional Drug Carrier for Tumor-Specific Uptake

milliliter of solution was collected from the flask periodically, and the released drug was determined spectrophotometrically. Volume of the release medium in the flask was kept constant by adding 1 mL of fresh medium after each sampling. Cell Culture. HepG2 cell, a human hepatocellular liver carcinoma cell line, was purchased from European Collection of Cell Cultures (ECACC), UK, and used for the cell uptake studies. The cells were grown in 75 cm2 culture flasks as a monolayer using Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, US) supplemented with 10% fetal bovine serum (FBS, Invitrogen, US) at 37 °C in 5% CO2, 95% humidified atmosphere in an incubator. The cells were subcultured every 48 h and, before testing, were harvested by trypsinization using a 0.05% (w/v) trypsin/0.03% (w/v) EDTA solution. Confocal Laser Scanning Microscopy Observation. HepG2 cells (2 × 105 cells/well) were cultured onto a plate with six wells; each was placed by a coverslip. They were maintained in DMEM media supplemented with 10% FBS in a humidified atmosphere of 5% CO2 at 37 °C for 24 h. The medium was then removed, and fresh DMEM was added at a desired pH, adjusted by adding 0.1 M aqueous HCl or NaOH, containing the drug-loaded polymeric micelles (5 wt %) with an equivalent doxorubicin concentration of 10 µg/mL. The cells were incubated for 0.5 to 2 h and were fixed using 4% paraformaldehyde for 30 min at room temperature. The coverslips were then mounted on the glass microscope slide with a drop of 80% glycerol PBS solution. The DOX distribution was observed using a confocal laser scanning microscope (CLSM, Leica TCSsp2, Germany) at excitation and emission wavelengths of 490 and 590 nm, respectively. Flow Cytometry. HepG2 cells (2 × 105) were maintained in DMEM supplemented with 10% fetal bovine serum for 24 h at 37 °C in 5% CO2, 95% humidified atmosphere on a 24-well plate. The medium was then changed by freshly prepared DMEM containing the drug-loaded polymeric micelles with an equivalent doxorubicin concentration of 10 µg/mL at pH 6.8 and 7.4. The cells were incubated for desired time, harvested and washed three times with PBS, and suspended with PBS. Flow cytometry was performed on a FACS calibur (BD Biosciences US). Hemolysis. Approximately 5 mL of porcine blood was poured into a centrifuge tube. PBS (pH 7.4) was added to 50 mL, and the solution was mixed. The tube was centrifuged (Heraeus Labofuge 400R Centrifuge) at 2500 rpm for 10 min at 4 °C. The erythrocyte pellet was isolated, washed twice with phosphate buffer by resuspending the pellet in PBS (pH 7.4), and centrifuged. The pellet was weighed, and 3 wt % dispersion of the erythrocyte was prepared in PBS. 80 µL of the erythrocyte suspension was pippetted into a 96-microwell plate, followed by adding 80 µL of polymer solution of different concentration in each well. PBS and Triton X-100 (1 vol %) served as the negative and positive control, respectively. The 96-microwell plate was incubated at 37 °C for 1 h and then centrifuged at 2500 rpm for 10 min at 4 °C. The absorbance of the supernatant (100 µL) was measured at 570 nm using a microplate reader (Versamax Tunable Microplate Reader). The results were expressed as percentage hemolysis with the assumption that Triton X-100 causes 100% hemolysis and PBS, 0% hemolysis. The above experiment was repeated at different pHs (6.8, 6.5, and 5.0).

RESULTS AND DISCUSSION Characterization of Polymer Structure and the pHDependent Hydrolysis of PEG-b-C18. The synthesis procedure of the PEG-b-C18 polymer is illustrated in Figure 2. The chemical structure of the polymer was examined using FTIR and 1H NMR in chloroform. In the NMR spectrum,

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Figure 2. Schematic description of the synthesis of the amphiphilic PEG-b-C18. Further reduction of imine bond in the PEG-b-C18 to amine gets the non-acid-labile PEG-C18.

the aldehyde peak at 9.7 ppm of PEG-CHO was not observed from the purified PEG-b-C18, while the imine proton at 8.1 ppm was clearly seen. The appearance of a specific band at 1648 cm-1 in the FTIR spectrum further confirmed the formation of the benzoic imine linkage in the PEG-b-C18 (see Supporting Information) (35, 36). The purification of the polymer was also checked by thin layer chromatography (TLC) on a silica plate. The hydrolysis kinetics as well as the hydrolysis bonding equilibrium of an imine bond is significantly influenced by environmental pH and the substitute groups (37, 38). While the imine bond formed from aliphatic amine and aldehyde hydrolyzes in neutral aqueous solution, the imine linkage in a Schiff base amphiphile made from aromatic amine and aldehyde is more stable due to the π-π conjugation (32-34). However, for the purpose of pH-triggered drug release, it requires an acid-labile bond in the carrier-building polymer responsive to a subtle pH difference under the physiological conditions, e.g., pH 4.5-7.4 (39, 40). Herein, we synthesized the amphiphilic polymer by an aromatic aldehyde (PEGbenzaldehyde) and an aliphatic amine (n-octadecane amine) for the formation of a derivate imine linker with proper conjugation extent. The stability and pH-dependent hydrolysis of the benzoic-imine bond in the PEG-b-C18 was monitored using 1H NMR, by dissolving the polymer in aqueous solution at different pHs (see Supporting Information). The aldehyde peak was not detected from the polymer in deuterated water at pH 7.4 for up to 24 h, indicating that the benzoic-imine bond is stable. At a pH of 6.5, the aldehyde peaks appeared where the imine peak still existed, an indication of partial hydrolysis of the polymer. By comparing the integrals of the aldehyde and imine peaks in the NMR spectra, it was shown that the hydrolysis extent of PEG-b-C18 increased with the decrease of solution pH. In addition, the hydrolysis reaction was rapid and reached equilibrium within 10 min, similar to our previous study (35). At a more acidic pH, i.e., 5.0, the

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Figure 4. Zeta potential of PEG-b-C18 (O) and PEG-C18 (•) micelles, and I3/I1 of pyrene fluorescence emission in PEG-b-C18 (9) and PEGC18 (0) solution as a function of solution pH. The polymer concentration is fixed at 0.5 mg/mL. pH range A, extracellular pH of solid tumor; B, endosomal pH.

Figure 3. TEM image (a) and DLS diagram (b) of PEG-b-C18 aggregates prepared at pH 7.4.

benzoic imine linker completely hydrolyzed, evidenced by the disappearance of the imine proton peak (8.1 ppm) accompanied by the recovery of the integral of the aldehyde proton at 9.7 ppm in the 1H NMR. FTIR of the PEG-b-C18 dried from a solution at acidic pH (∼5.0) further confirmed hydrolysis of the imine linkage by the comparison of the specific band at 1648 cm-1 with that of the untreated sample. Aggregate Formation and pH-Mediated Structural Evolution of PEG-b-C18. Micelles were formed from the PEG-bC18 in aqueous solution at neutral and basic pH. The cmc was determined as 0.019 mg/mL at pH 7.4 using fluorescence spectrophotometry with pyrene as the probe. The aggregates are spherically shaped in the TEM image (Figure 3a) and have an average size of 181 nm measured by DLS (Figure 3b), implying the formation of a multicore structure through the entanglement of the PEG chains (41). The pH dependence of zeta potential and the core polarity of the aggregates, determined by fluorescence emission of pyrene, were plotted in Figure 4. At a pH higher than 7.4, the I3/I1 of the PEG-b-C18 micelles is larger than 1 and the zeta potential is nearly zero, because the benzoic imine linker is stable; thus, the micelles contain no charged moiety. A sharp increase of zeta potential from 0 to 30 mV is observed with the pH decrease from 7.0 to 6.5 for the PEGb-C18, attributed to the cleavage of PEG chains generating positively charged octadecane amine (pKa ∼11), whereas the zeta potentials of the nondegradable PEG-C18 micelles are nearly constant at different pHs (Figure 4). However, since the I3/I1 ratio of pyrene emission remains at a higher value, i.e., 1.15, the micellar structure should be maintained for the PEG-b-C18 at pH 6.5-5.0, while the zeta potential reaches a plateau at 30 mV. The average particle size of the PEGb-C18 micelle at pH 6.5 is 183 nm (DLS), almost the same as the original 181 nm at pH 7.4. As described above, an acidic pH higher than 5.0 would not cause a complete cleavage of the benzoic-imine bond. It is understandable that the portion of uncleaved PEG-b-C18 molecules prevented

Figure 5. Cumulative release of doxorubicin from the PEG-b-C18 micelles at pH 7.4 (9) and 6.8 (•) and from the PEG-C18 micelles at pH 7.4 (0) and 6.8 (O).

the micellar structure from dissociating, and the residual octadecane amine from the cleaved polymer was solubilized in the core of the micelle with amino groups exposed on the surface leading to a positive zeta potential. The transition pH of the zeta potential is 6.7, defined as the middle value between the two inflections of the curve (Figure 4), lying in the extracellular pH region of solid tumors. The positively charged surface of the PEG-b-C18 micelles will be conducive for cell absorption in the tumor site (10). At a lower pH, i.e., the endosomal pH ∼5.0, further cleavage of the PEG-b-C18 will cause dissociation of the micelles. As shown in Figure 4, the I3/I1 ratio of the PEG-b-C18 decreases abruptly to a value close to that for water, i.e.. 0.7, an indication of demicellization (42). The fluorescence intensity was also reduced significantly at pH 5.0 due to the decrease of the quantum yield and the quenching of fluorescence by the primary amino group of the produced octadecane amine (43). Thus, with the environmental pH changing from physiological to tumor and endosomal pHs, the progressive hydrolysis of the benzoicimine bond in the PEG-b-C18 leads to a surface property change and furthermore a structural change of the aggregates, i.e., from PEG-shielded to positively charged and finally dissociated (Figure 4).

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Figure 6. Confocal fluorescence microscopy images (in each column: left, doxorubicin channel; right, overlap of doxorubicin and transmittance channels) of HepG2 cells being treated with free DOX, DOX-loaded PEG-b-C18 micelles, and DOX-loaded PEG-C18 micelles with incubation time of 0.5 and 2 h at pH 7.4 and 6.8.

Drug Loading and in Vitro Release. Doxorubicin was encapsulated in the micelles using the solvent evaporation method. The PEG-b-C18 micelle with a drug loading capacity of 30 wt % has a mean size of 216 nm (DLS), slightly bigger than that of the bare micelle (181 nm). The size increase is attributed to the solubilization of drug within the hydrophobic core. Figure 5 shows the DOX release from the PEG-b-C18 and the PEG-C18 micelles at different pHs. A burst release is observed at early stage of the profiles, which may be explained by the relatively low molecular weight of the polymer especially for the hydrophobic segment, leading to a lower stability of the entrapped drug molecules in the hydrophobic cores (1). Nevertheless, by comparison with the PEG-C18 micelles, it could be clearly seen that the release of DOX from the PEG-b-C18 micelles was accelerated by changing the environmental pH from neutral to slightly acidic, i.e., 7.4 to 6.8 (Figure 5). Similar pH dependence on the release of prednisolone, a drug with constant water solubility in the tested pHs, from the PEG-bC18 micelles was also observed, indicating that the PEG-bC18 formulations are able to respond to a very subtle change of the environmental pH. In Vitro Cellular Uptake of DOX-Loaded PEG-b-C18 Micelles at Tumor and Physiological pHs. The intracellular localization of DOX-loaded PEG-b-C18 micelles was investigated using confocal fluorescence microscopy on HepG2 cells (Figure 6). As expected, DOX is mainly distributed in the nuclear region of cells after being treated with free drug solution (9, 26). Differently, the fluorescence intensity was detectable in both nucleus and cytoplasm after the cells were incubated with the DOX-loaded PEG-b-C18 micelles. This proves that the internalization mechanism of the PEG-b-C18 micelle is endocytosis (44). However, the amount of DOX uptake showed strong pH dependence for the DOX-loaded PEGb-C18 formulations. Flow cytometry shows that the mean fluorescence intensity is much higher in the cells treated with the drug-loaded PEG-b-C18 micelles at pH 6.8 than that at physiological pH 7.4, whereas it has little difference and stays

at a low level after treatment with the noncleavable PEG-C18 formulations at the two pHs (Figure 7). The internalization of noncharged nanoparticles, for example, with a PEGylated surface, normally undergoes a fluid-phase endocytosis mechanism, while positively charged nanoparticles will be taken up by cells through a more efficient adsorptive endocytosis due to the electrostatic interaction between the carrier and the negatively charged cell membrane (45). However, both of the processes are nonspecific. In our studied system, the hydrolysis of the benzoic-imine bond in PEG-b-C18 occurs at tumor pH and the micelle surface converts from neutral to positively charged with the micellar structure preserved (Figure 4). Therefore, the adsorptive endocytosis is favored only at the tumor pH (6.8) to the PEG-b-C18 micelles, which would lead to the acceleration of DOX uptake under the tumor condition. pH-Dependent Membrane Disruption of PEG-b-C18. After being endocytosed, a rapid endosomal escape of the carrier is required for protecting the carried drug from degradation during the lysosome transport. The pH-dependent membrane disruption of the micelle from physiological to endosomal pH was evaluated using porcine erythrocytes as the biological model membrane (22, 46). Under physiological and tumor conditions (pH ) 7.4-6.5), hemolysis of the PEGb-C18 micelle increases slightly with the decrease of pH (Figure 8). This proves again that the positive ionization of the PEG-b-C18 micelle at tumor pH indeed increases the electrostatic interaction between the micelle and the cell membrane. The hemolysis is 22% at pH 7.4 with a polymer concentration of 10 mg/mL, in a reasonable range for intravenous administration, and remains at relatively low levels (