Polymeric Micelles with Uniform Surface Properties and Tunable Size

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Polymeric Micelles with Uniform Surface Properties and Tunable Size and Charge: Positive Charges Improve Tumor Accumulation Tong Shen, Shuli Guan, Zhihua Gan, Guan Zhang, and Qingsong Yu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00234 • Publication Date (Web): 23 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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Polymeric Micelles with Uniform Surface Properties and Tunable Size and Charge: Positive Charges Improve Tumor Accumulation Tong Shena, Shuli Guana, Zhihua Gana, Guan Zhangb and Qingsong Yua a

The State Key Laboratory of Organic-inorganic Composites, Beijing Laboratory of

Biomedical Materials, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, China b

Department of Urology, China-Japan Friendship Hospital, Beijing 100029, China

Keywords: Micelle size; surface charge; uniform surface property; tumor accumulation; cellular uptake

1

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ABSTRACT. The influences of surface charge on the biodistribution and tumor accumulation remain debatable because most of the researches were carried out by changing the surface functional groups of nano-carriers. In this work, to avoid the interference of different surface properties such as chemical composition and hydrophilicity, polymeric micelles with uniform PEG coatings and continuously tunable size or zeta potential were developed via a facile route. Therefore, the influence of surface charge on the biological functions of micelles with same size and surface property could be well explored. In this case, positive charge was found to enhance both tumor cellular uptake and tumor accumulation. Immunofluorescent staining indicated that the improved tumor accumulation was mainly due to the tumor vasculature targeting of positively charged micelles. It is predictable that the efficient drug delivery systems with both tumor vasculature and cancer cells targeting could be realized based on positively charged micelles.

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1. Introduction In order to prevent undesirable side effects exercised onto normal cells, organs and tissues by cytotoxic drugs, and to increase drug bioavailability and the fraction of the drug accumulated in the pathological area, various types of nanoparticles (NPs), including liposomes, polymeric micelles, albumin-based particles, have been widely used for controlled release of anti-cancer drugs.1-8 Among them, polymeric micelles from amphiphilic block copolymers received great attentions since they can self-assemble into core-shell structures with controllable size, charge and surface morphology.9, poly(ethylene

10

Micelles assembled from amphiphilic copolymers such as glycol)-b-polycaprolatctone

(PEG-b-PCL),11

poly(ethylene

glycol)-b-polylactide (PEG-b-PLA),12 and PEG-b-polypeptide13 showed prolonged blood circulation time and improved tumor accumulation through the enhanced permeability and retention (EPR) effect.14 It has been reported that physicochemical characteristics such as particle size and surface charge have significant impacts on the biological functions of drug delivery systems (DDSs) by influencing the adhesion of the particles and their interaction with cells.15 For the impact of particle size, the consensus is that, under the premise of the blood circulation is not affected, large particles can accumulate in tumor more efficiently due to the EPR effect. Even though small particles exhibited lower tumor accumulation, they can penetrate much deeper into the tumor tissue than large ones, especially in poorly permeable tumor models. Therefore, there are complex 3


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dependencies between the eventual tumor inhibition outcome and particle size.16, 17 In contrast to particle size, the influence of surface charge on the biological functions of various DDSs remains inconsistent.18-20 Early work based on the liposomes have clarified the possibility of positive charged liposomes to target the tumor vasculature. With positive charge, liposomes can be detained in tumor for a longer time than neutral or negatively charged ones.2,

21-25

However, similar

phenomena have merely been reported for any other systems. A report covering the systematic distribution and clearance of protein with different charge hold that a drug-carrier complex designed for systemic tumor targeting should be polyanionic in nature.26 Recent researches about polymer drug conjugates in author’s group have reached the similar conclusion except that only slight amount of negative charge is beneficial for prolonging the blood circulation and tumor accumulation.27, 28 Another research about neutral and negatively charged PEG-b-PLA micelles demonstrated that the non-specific uptake by liver and spleen was significantly suppressed due to the electrostatic repulsion between negatively charged micelles and cell membrane.29 Besides the above report, Xiao et al.30 reported a series of micellar nanoparticles based on the distal PEG termini of monomeric PEG-oligocholic acid dendrimers. The results showed that undesirable liver uptake was high for both highly positively and highly negatively charged micelles. On the contrary, for micelles with slightly negative charge, low liver uptake and high tumor accumulation was observed. Another work reported by He et al.31 exhibited the cellular uptake and biodistribution of 150 nm and 500 nm chitosan nanoparticles with different surface charge. The in 4


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vivo biodistribution suggested that 150 nm nanoparticles with slightly negative charge tend to accumulate in tumor more efficiently. In summary, most works have emphasized the typical characteristics of positive charge such as the increased interaction with erythrocytes and mononuclear phagocytic system (MPS) which lead to elevated lung, liver and spleen accumulation.30-32 Superficially, it could be concluded from the above researches that positive charge might not be beneficial for the efficient delivery of anticancer drugs. However, when carefully studied these past literatures, one can find that the regulation of charge was mainly realized by changing the surface groups of DDSs. This will inevitably influence the consistency of the surface chemical composition as well as the accuracy of the results. Therefore, the major concern should be how to maintain the surface properties of nanoparticles while altering their size or surface charge. Herein, based on the previous reported poly(ethylene glycol)-block-poly (N-methacryloyl-N′-(t-butoxycarbonyl) cystamine) (PEG-b-PMABC) copolymers,33 the authors proposed a new way to prepare micelles with different size or surface charge by altering the charge of the inner core. Under this circumstance, the micelle surface will always be protected by hydrophilic chains such as PEG; and the consistency of the surface properties could be well maintained. Based on the consistent surface properties, the preliminary in vitro and in vivo fates of polymeric micelles with different size and surface charge were investigated.

2. Material and methods 2.1. Materials 5


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Poly(ethylene glycol) monomethyl ester (MPEG-OH) (Mn=5000, Mw/Mn=1.05), cystamine dihydrochloride, di(tert-butyl) dicarbonate, N-hydroxysuccinimide (NHS), dicyclohexylcarbodiimide

(DCC),

4-dimethylaminopyridine N-(3-aminopropyl)

N-t-butoxycarbonyl-ethylenediamine, hydrochloride

(APMA),

doxorubicin

(DMAP),

methacrylamide

hydrochloride

(DOX),

rhodamine-B-isothiocyanate (RBITC), trifluoroacetic acid (TFA), acetic acid (HAc) and 4,4′-azobis(4-cyanopentanoic acid) (V501) were obtained from Sigma-Aldrich and used as received without further purification. A lipophilic near infrared cyanine dye,

Cy7.5,

was

obtained

Dithioester-capped

PEG

N-methacryloylglycylglycine cystamine

from

Lumiprobe

(PEG-CTA), (MAGG),

(MABC),

(Hallandale

Beach,

FL).

N-(t-butoxycarbonyl)cystamine,

N-methacryloyl-N′-(t-butoxycarbonyl)

N-methacryloylglycylglycyl-N’-t-butoxycarbonyl

ethylenediamine (BEMAGG), PEG-b-PMABC and PEG-b-PBEMAGG were prepared according to the literatures.33, 34 Rhodamine B labeled monomer MA-RBITC was prepared following the method previously reported by the author’s group by simply

changing

the

fluorescein

isocyanate

(FITC)

with

RBITC.34

The

PEG-b-PMABC-b-PS tri-block copolymers were synthesized by the sequential polymerization of MABC and styrene monomers with the same method as PEG-b-PMABC. The Cy7.5 labeling of tri-block copolymers was carried out according the our previous reports.28 2.2. Characterization The 1H NMR spectra of the compounds were characterized on a Bruker FT-NMR 6


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spectrometer operating at 400 MHz at room temperature. The molecular weight and molecular weight distribution (Mw/Mn) of polymers were determined by gel permeation chromatography (GPC). The GPC measurement was performed by waters 1515 GPC system with THF as eluent and a flow rate of 1.0 mL/min at 40 °C. Polystyrenes with narrow distributed molecular weights were used as standards for calibration. Transmission electron microscopy (TEM) was performed using a JEOL JEM-1200EX TEM at an accelerating voltage of 200 kV. The TEM samples were prepared by first placing a drop of 10 µL micelles solution (0.5 mg/mL) onto a formvar/carbon coated 200 mesh copper grid. After 5 min, the excess solution was wicked off by using filter paper. Then the grid was left to dryness under ambient conditions. Dynamic light scattering (DLS) was performed with a Malvern Zetasizer Nano ZS instrument. He-Ne laser (633 nm wavelength) with a fixed detector angle of 173° was used for the measurements. All measurements were carried out at 25 °C. 2.3. The preparation of micelles Micelles were prepared by a dialysis method. Briefly, 1.0 mL of block copolymer solution (10 mg/mL) in water-miscible solvents was added dropwise into distilled water (10.0 mL). The solution was stirred for 2 h before transferred into a dialysis tube (Mw cutoff, 7000 Da). The solution was then dialyzed against distilled water for 3 days with water changed every 12 h. The preparation of drug loaded micelles is similar to normal micelles except adding 5 mg anticancer drug doxorubicin (DOX) and 50 µL triethylamine in copolymer solution. The drug loading content (DLC) was determined by high performance liquid 7


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chromatography with Acetonitrile/water (v/v=1/1) as elute. The flow rate was set as 1 mL/min. To prepare the three layer tri-block polymeric micelle, a given amount of PEG114-b-PMABC15-b-PS33 was dissolved in 0.2 mL THF or DMF and then added to 1 mL DMSO to form the inner core with PS block. After that the DMSO solution was added dropwise to 10 mL water to allow the formation of the middle layer with PMABC block. The solution was then dialyzed against distilled water for 3 days with water changed every 12 h. The micelle size could be adjusted by the polymer solution as well as the block length. Fluorescently labeled tri-block micelles were prepared with Cy7.5 or rhodamine labeled tri-block copolymers using the same method. 2.4. Deprotection of Boc-protected amino groups A given amount (2-24%, V/V) of TFA was added into micelles solution and the solution was stirred at room temperature for 0-48 h. Then NaOH (10 mM) was added into the solution to halt the deprotection procedure. The solution was transferred into a dialysis tube (cellulose membrane, Mw cutoff: 7000 Da) and dialyzed against distilled water for 3 days with water change every 12 h to remove TFA and other impurities until the pH of solutions reached 6.8-6.9. The amount of TFA and the reaction time were varied to control the degree of deprotection which lead to the change of size and surface charge of micelles. For deprotection in DMSO, PEG-b-PMABC was firstly dissolved in DMSO. Then TFA was added to remove the Boc group for predetermined time. After deprotection, 10 mM NaOH was added to neutralize TFA. Then the DMSO solution was added 8


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dropwise into distilled water to form micelles. The resultant salts were removed by dialysis method. 2.5. Cellular uptake of drug-loaded micelles Henrietta Lacks strain of cancer cells (Hela) were cultured in 1640 Medium supplemented with penicillin, streptomycin and 10% fetal bovine serum (FBS). Cells were maintained at 37 °C in a humidified atmosphere of 5% carbon dioxide and 95% air. All tissue culture media were obtained from Gibco Life Technologies, Inc. (Grand Island, NY). For the analysis of cellular uptake by Leica TCS-SP8 Confocal Laser Scanning Microscope (CLSM), Hela cells were plated in glass bottom petri dish 12 h prior to the experiment. Cells were treated with drug-loaded micelles with 10 µg/mL DOX concentration and incubated for a certain time at 37°C /5% CO2. Before CLSM observation, cells were washed subsequently thrice with ice-cold PBS, and the cell nucleus were stained by Hoechst 33342. For the analysis of cellular uptake by flow cytometry, Hela cells were plated in 6-well plates (1×105 per well) 1 day prior to the experiment. Cells were then treated with drug-loaded micelles at 10 µg/mL DOX concentration and incubated at 37°C /5% CO2, washed subsequently thrice with ice-cold PBS, trypsinized and centrifuged. The cell pellet was resuspended in 400 µL PBS and then analyzed using flow cytometry. 2.6. Cytotoxicity assay Hela cells were plated in 96-wall plates and treated with drug-loaded micelles or free DOX at 10 µg/mL DOX equivalent for a certain time. Cell viability after 9


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treatments by different micelles was measured by Cell Counting Kit-8 (CCK-8) assay with microplate reader (Multiskan GO, Thermo Scientific). 2.7. In vivo fluorescent imaging of polymer-drug conjugates. All animal experiments were performed under a protocol approved by Institutional Animal Care and Use Committee of Peking University. Five-week-old female BALB/c mice were fasted for 6 h before the experiment to eliminate disturbing signal and obtain comparable results. To obtain tumor-bearing mice, each BALB/c mouse was subcutaneously injected 1×106 4T1 cells. Solid tumors were allowed to form about 1 week to reach a volume ranging from 50 to 100 mm3. Then the mice were anesthetized by isoflurane and placed flat on the stomach. Optical imaging of the fluorescein-loaded micelles in tumor-bearing mice was conducted on an IVIS® Spectrum in vivo imaging system (Perkin Elmer, U.S.A) using 745/800 filter combination. The photos were acquired and analyzed by Living Image version 4.4 In Vivo Imaging Software (Perkin Elmer, U.S.A). The exposure time, F/stop and Binning values were set as 5s, 1 and 4, respectively. The images were intensity weighted and displayed using the rainbow color profile. Blood clearance analysis was carried out by retro-orbital bleeding technique with 3 mice in each group at predetermined time intervals. 2.8. Immunofluorescent staining. For CD31 staining of tumor vasculature, tumor frozen sections were incubated overnight at 4°C with the Pierce™ CD31 / PECAM-1 antibody (Thermo Fisher Scientific). After being washed and incubated with protein-blocking solution, they 10


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were incubated for 1 h at room temperature with Goat anti-Rabbit IgG (H+L) Secondary Antibody (Thermo Fisher Scientific) which was conjugated to Alexa Fluor® 488 as previously described in Ref.35 DAPI was used to stain the cell nucleus. Fluorescent images were acquired with a Leica DMI 3000B fluorescent microscope and processed with the Image Pro Plus software (Media Cybernetics, Inc., Silver Spring, MD). 2.9. Statistic analysis Comparison between two groups of data was made using the one-way ANOVA test (Graph Pad Prism 5.0, San Diego, CA). The level of significance was set at p=0.05.

3. Results and discussion 3.1. Preparation and characterization of polymeric micelles In order to accurately disclose the direct impact of charge on the biological properties of polymeric micelles, one need to make sure that the size and surface properties maintain consistent. For this consideration, amphiphilic block copolymers PEG-b-PMABC and PEG-b-PBEMAGG as reported previously by author’s group33 were introduced to evaluate the possibilities of fabricating polymeric micelles with controllable size and surface charge. The common features of the two types of copolymers are the Boc protected amino groups and the PEG hydrophilic layer. Different from the traditional way of changing surface functional groups, the regulation of micelles’ charge was realized by the deprotection of Boc groups in the inner core. Therefore, the surface properties should maintain the same during the deprotection process. 11


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The synthetic routes of copolymers used in this work could be found in Figure 1A. Both the GPC (Figure 1B & D) and 1H NMR results (Figure S1) have clearly indicated the well-controlled structure and composition. Micelles based on block copolymers were prepared by dialysis method. Figure 1C shows the typical size distribution of the 1 mg/ml PEG114-b-PMABC37 micelles prepared with THF, DMSO and DMF, respectively. The inset picture represents the typical TEM graphs of the micelles prepared with THF. The narrow size distribution (~0.16) could be attributed to the well-defined polymer structure. The micelle size exhibited an obvious solvent dependence, that is, DMF < DMSO < THF. This was in accordance with the literature reports.16 Although the reason for this phenomenon was still unclear by now, the author inferred that it might be the different inter- or intramolecular hydrogen bond strength of PEG-b-PMABC block copolymers in various solvents. Based on these solvent effects, much finer hydrodynamic size regulation could be realized by tuning parameters such as molecular weight, polymer concentration as well as the volume ratio of organic solvents to water.

12


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Figure 1. (A) Synthetic routes of di-block and tri-block copolymers. (B) GPC elution curves of PEG114 macro CTA and PEG114-b-PMABC37 block copolymers. (C) Size distribution of PEG114-b-PMABC37 micelles prepared with DMF, DMSO and THF, respectively. The inset picture represents the typical TEM graph of micelles prepared with THF. (D) GPC elution curves of tri-block copolymers. (E) Size distribution of tri-block polymer micelles with different size. The inset picture represents the typical TEM graph of 65 nm micelles.

3.2. Adjustment of micelle size and surface charge To evaluate the possibility to control micelles’ charge with TFA, a stepwise deprotection method was introduced. After the addition of TFA, Boc groups will be removed gradually so as to expose the amino groups. Figure 2 represents the 1H NMR spectra of polymeric micelles in deuterium oxide before (A) and after (B) deprotection. Only the signals from PEG block (3.63 ppm from -CH2CH2O-) can be detected at the beginning due to the hydrophobic nature of PMABC block (Figure 2A). However, after being treated with TFA for 48 hours, Boc groups were removed and the former hydrophobic part became hydrophilic. That was why the NMR signals at 3.31 ppm, 2.87-2.93 ppm, and 1.22 ppm from PMABC block could be detected in Figure 2B.

13


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Figure 2. The 1H NMR spectra of PEG114-b-PMABC37 micelles in D2O before (A) and after (B) treating with 2 % deuterated TFA for 48h. (C) The size and Derived Count Rate alteration with the increase of TFA concentration (each point was measured at 48h after TFA treatment). (D) The final zeta potential values of di-block micelles treated with TFA in water and DMSO, respectively.

The stability of micelles in different TFA concentration was showed in Figure 2C as the alteration of micelle size and scattering light intensity (Derived Count Rate (DCR)). It can be found that the same turning points existed (CfTFA=8%) in both curves. When the acid concentration was lower than 8%, both the size and scattering light intensity increased slowly with the acid concentration. When the acid concentration was higher than 8%, the micelle size underwent a drastic increase, but the DCR which represents the scattering light intensity is known to be dependent on 14


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both the particle size and particle number (Malvern Zetasizer Nano Series User Manual) decreased sharply. This indicated that the micelles collapsed at high TFA concentration and larger aggregates formed thereafter. Therefore, all the experiments in this work were carried out with acid concentration lower than 8%.

To explore the deprotection process, the block copolymers were treated with TFA in water and DMSO, respectively. The TFA treating time was set as 48 h for both solvents so that the deprotection was completed. Since PEG-b-PMABC can be completely dissolved in DMSO, the deprotection in TFA/DMSO solution was regarded to be homogeneous and randomized. The dependence of equilibrated zeta potential on the TFA concentration could be found in Figure 2D. The general trends of zeta potential alteration with TFA concentration were in consistency for polymers treated with TFA in both water and DMSO. The difference lied in the final zeta potential values. In water, the addition of TFA with concentration as low as 2% caused a drastic charge conversion from -20 mV to +38 mV. However, the same TFA concentration in DMSO only caused a slight zeta potential change from -20 mV to -10 mV. The highest TFA concentration (8%) brought a final zeta potential value of +55 mV for polymers deprotected in water while only +8 mV for polymers treated in DMSO. As mentioned above, the removal of Boc groups was randomized in DMSO, therefore, the amino groups were thought to be randomly dispersed on the polymer chain. When these polymers formed micelles in water, the movement of amino groups was restricted by their adjacent hydrophobic Boc groups so that their positive charge cannot be efficiently demonstrated. The efficient charge conversion in water 15


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suggested that the deprotection was in an ordered manner from outside to inside instead of a random process. The typical size and zeta potential change of di-block micelles prepared in different solvents after treated by different concentration of TFA could be found in Figure S2; and the size distribution during the deprotection process could be found in Figure S3. Intriguingly, micelle size increased immediately right after the addition of TFA and then kept almost unchanged during further incubation time. Besides, micelle size was closely related to the acid concentration, that is, more TFA resulted in larger size. In contrast to the size, zeta potential has been increasing during the whole incubation period and reached the plateau at about 48 h. The 1H NMR spectra of micelles treated by different amount of deuterated TFA could be found in Figure S5. With the increase of TFA concentration, the integration area of methyl groups at 1.2 ppm increase accordingly, indicating more MABC exposed on the hydrophilic corona. In order to find out the reason for the sudden change of micelle size after the addition of TFA, micelles were treated with TFA and HAc, respectively. Under this circumstance, HAc is too weak to deprotect Boc groups. Figure 3 illustrates the possible deprotection mechanism of polymeric micelles in water. It could be observed that the micelle size underwent a sudden increase after the addition of both TFA and HAc. As reported in literature,36 the amide group could be protonated under the condition of TFA which make MABC monomer to be partially hydrophilic. The authors supposed it might be one of the reasons, the other reason might be the alteration of intra- or inter-molecular hydrogen bonds. To prove this hypothesis, the 16


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TFA or HAc treatment was quenched immediately by dilute NaOH solution right after the addition of the acid and then dialyzed against distilled water to remove impurities. Then the micelles were subjected to light absorbance measurement at 500 nm. As shown in Figure S4, the temperature-dependent light absorbance transition could be observed for both TFA and HAc treated micelles. On the contrary, no transition could be observed for untreated micelles. Even though the reason for this transition temperature change was still unclear, it indirectly indicated the alteration of hydrogen bonds inside micelles. Moreover, with the prolonging of incubation time, TFA can remove the Boc groups much more efficiently than HAc. That is why the zeta potential of TFA treated micelles increased gradually with time while that of HAc treated micelles maintained unchanged. Besides, it was noteworthy that the zeta potential could be fixed at any predetermined value by quenching the deprotection at appropriate time. This can further prove that the initial size increase was not caused by the deprotection of Boc groups.

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Figure 3. Schematic illustration of the deprotection procedure and the size and zeta potential change with time for di-block or tri-block micelles treated with TFA or HAc.

The above results provided us with a facile way of fabricating polymeric micelles with predetermined size and zeta potential. However, considering the partially hydrophilic nature of the protonated MABC block, as-prepared micelles might not be stable enough during the blood circulation. To ensure the stability, PS block was introduced to provide a stable core as reported in literature.37 The synthesis and characterization of tri-block copolymers could be found in Figure 1A & D. Three layer polymeric micelles based on tri-block copolymers with different size were prepared (Figure 1E). When the di-block copolymers were substituted with tri-block copolymers, no sudden change of micelle size could be observed after adding TFA due to the restriction of PS core due to its high Tg (80~105°C);37 but the tendency of 18


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zeta potential increase was similar to that of di-block copolymers. Under this circumstance, tri-block micelles with different size and surface charge could be obtained by TFA treatment of micelles with different initial size for predetermined time. To test their stability in blood circulation, tri-block micelles with various size and zeta potential were co-incubated with 50% fetal bovine serum at 37°C. As shown in Figure S6, micelle size did not undergo any change for micelles with different charge during 72 h incubation period. This could be interpreted from two aspects, on one hand, it indicated the stability of as-prepared micelles in blood circulation; on the other hand, it also reflected the consistent surface properties of micelles with different charge. 3.3. Cellular uptake and cytotoxicity of di-block polymeric micelles with different zeta potential Table 1. Physicochemical characterization of di-block or tri-block polymeric micelles Sample

Size (nm)a

Polymer

Zeta potential (mv)

a

a

DLC

TFA

Deprotection

(%)

concentration

time (h)

M1

PEG114-b-PMABC37

118.3±1.4

-11.7±1.1

12.9

2%

0

M2

PEG114-b-PMABC37

123.1±2.1

0.2±0.9

12.8

2%

2

M3

PEG114-b-PMABC37

125.7±1.9

6.4±1.5

12.8

2%

4

B1

PEG114-b-PBEMAGG33

119.6±2.3

-12.7±1.3

12.4

2%

0

B2

PEG114-b-PBEMAGG33

128.5±1.2

-1.1±1.5

12.1

2%

2

B3

PEG114-b-PBEMAGG33

132.3±1.5

7.2±1.3

12.1

2%

4

65 (-)

PEG114-b-PMABC15-b-PS38

64.8±1.1

-10.1±0.8

-

4%

1

65 (0)


65.3±1.2

0.2±1.2

-

4%

6

65 (+)


65.1±1.5

8.1±1.9

-

4%

24

96 (-)


95.1±1.9

-9.6±2.1

-

4%

1

96 (0)


97.2±2.1

0.3±1.1

-

4%

6

96 (+)


98.4±1.6

7.2±1.6

-

4%

24

Size and zeta potential were obtained from DLS measurement in 10 mM PBS buffers (pH = 7.4).

Micelle concentration was set as 0.5-1.0 mg/mL.

19


Biomacromolecules

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Due to their special fabrication process, tri-block micelles are not suitable for the physical entrapment of anticancer drugs. Therefore, to investigate the influence of surface charge as single variable on the cellular uptake and cytotoxicity of drug loaded polymeric micelles, di-block polymeric micelles with similar size and different zeta potential values were prepared. The anticancer drug DOX was loaded into micelles by a simple dialysis method. The drug loading content (DLC) after TFA treatment were approximately 12 wt.% for micelles formed from PEG114-b-PMABC37 (M1, M2 and M3) and PEG114-b-PBEMAGG33 (B1, B2 and B3), respectively. Herein, PEG114-b-PBEMAGG33 micelles with similar size were introduced to evaluate whether disulfide bond interfere the micelle cell interaction of PEG114-b-PMABC37. The physicochemical characterization of micelles was listed in Table 1. It could be found that both PEG114-b-PMABC37 and PEG114-b-PBEMAGG33 micelles with different zeta potential showed similar size. This guaranteed that the surface charge was a single variable in the cellular evaluation of both micelles.

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Biomacromolecules

Figure 4. (A) CLSM images of Hela cells incubated with M1, M3 and free DOX at 37 °C. (B) Cellular uptake of DOX, B1, M1, M2 and M3 with the increase of time (*p

Polymeric Micelles with Uniform Surface Properties and Tunable Size

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