Zwitterionic-Modified Starch-Based Stealth ... - ACS Publications

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Zwitterionic-Modified Starch-Based Stealth Micelles for Prolonging Circulation Time and Reducing Macrophage Response Lei Ye,† Yabin Zhang,† Boguang Yang,† Xin Zhou,‡ Junjie Li,∥ Zhihui Qin,† Dianyu Dong,† Yuanlu Cui,*,‡ and Fanglian Yao*,†,§ †

School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, China Tianjin State Key Laboratory of Modern Chinese Medicine, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, China § Key Laboratory of Systems Bioengineering of Ministry of Education, Tianjin University, Tianjin 300072, China ∥ Department of Advanced Interdisciplinary Studies, Institute of Basic Medical Sciences and Tissue Engineering Research Center, Academy of Military Medical Science, Beijing 100850, China ‡

S Supporting Information *

ABSTRACT: Over the last few decades, nanoparticles have been emerging as useful means to improve the therapeutic efficacy of drug delivery and medical diagnoses. However, the heterogeneity and complexity of blood as a medium is a fundamental problem; large amounts of protein can be adsorbed onto the surface of nanoparticles and cause their rapid clearance before reaching their target sites, resulting in the failure of drug delivery. To overcome this challenge, we present a rationally designed starch derivative (SB-ST-OC) with both a superhydrophilic moiety of zwitterionic sulfobetaine (SB) and a hydrophobic segment of octane (OC) as functional groups, which can self-assemble into “stealth” micelles (SSO micelles). The superhydrophilic SB kept the micelles stable against aggregation in complex media and imbued them with “stealth” properties, eventually extending their circulation time in blood. In stability and hemolysis tests the SSO micelles showed excellent protein resistance properties and hemocompatibility. Moreover, a phagocytosis test and cytokine secretion assay confirmed that the SSO micelles had less potential to trigger the activation of macrophages and were more suitable as a drug delivery candidate in vivo. On the basis of these results, doxorubicin (DOX), a hydrophobic drug, was used to investigate the potential application of this novel starch derivative in vivo. The results of the pharmacokinetic study showed that the values of the plasma area under the concentration curve (AUC) and elimination half-life (T1/2) of the SSO micelles were higher than those of micelles without SB modifications. In conclusion, the combination of excellent protein resistance, lower macrophage activation, and longer circulation time in vivo makes this synthesized novel starch derivative a promising candidate as a hydrophobic drug carrier for long-term circulation in vivo. KEYWORDS: starch, micelle, drug release, long-term circulation, zwitterionic

1. INTRODUCTION In recent years, nanocarriers have been widely investigated as drug delivery carriers for improving therapeutic effects and reducing side effects, owing to their multifunctional properties.1−3 Most researchers have focused on the special microenvironments of tumor sites and have fabricated sophisticated nanoparticles to improve therapeutic effects. However, it is well-known that the blood is a complex electrolyte medium containing oxygen, carbon dioxide, blood cells, and large amounts of proteins in particular. Consequently, nanoparticles that immediately enter into the blood are prone to adsorb a large amount of plasma proteins.4−6 These proteinmarked nanoparticles are recognized by the mononuclear phagocyte system (MPS), subsequently leading to their clearance by the MPS.7 Thus, a necessary requirement for © 2016 American Chemical Society

intravenous use of nanoparticles is their longer circulation time in blood in an extremely stable manner. Such nanoparticles must possess a “stealth” surface coating to maintain stability in complex media and minimize opsonization in blood. Furthermore, the nanoparticles must have the ability to bypass various clearance mechanisms in vivo, such as renal clearance,8 to remain in blood. Therefore, “stealth” properties play a paramount role in drug delivery. Until now, nonionic poly(ethylene glycol) (PEG) has been widely investigated to modify the surface of nanoparticles to achieve “stealth” properties.9−11 Studies suggest that a water Received: November 9, 2015 Accepted: February 2, 2016 Published: February 2, 2016 4385

DOI: 10.1021/acsami.5b10811 ACS Appl. Mater. Interfaces 2016, 8, 4385−4398

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ACS Applied Materials & Interfaces

Scheme 1. Scheme of the Synthesis and Self-Assembly of the Starch Derivatives: (A) Diagram of the Synthetic Pathway to Generate SB-ST, ST-OC, and SB-ST-OC and (B) Graphical Representations of the Self-Assembly Processes of SO and SSO Micelles

drates, exhibits biodegradability, nontoxicity, and nonimmunogenicity and is used extensively in the pharmaceutical field as an excipient for drug delivery, making it an ideal alternative to synthetic polymers for drug delivery.28,29 Traditionally, starch-based micelles have been prepared via the self-assembly of starch derivatives synthesized by the simple grafting of hydrophobic side chains to starch backbones.30−32 In this work, a series of starch-based micellar polymers containing both superhydrophilic zwitterionic (SB) and hydrophobic (OC) groups were prepared and characterized, called SB-ST-OC micelles (SSO micelles), with the aim of shielding the micelles from blood protein adsorption for longer blood residence times in vivo. As a control, a starch-based micellar polymer with only a hydrophobic (OC) group, called ST-OC micelles (SO micelles), was also prepared and characterized. The micelles were investigated by transmission electron microscopy (TEM), malvern zetasizer measurement, and fluorescence techniques. Interactions between proteins and the micelles were evaluated by measuring the particle sizes during their incubation in bovine serum albumin (BSA) solution and human serum. The adsorbed proteins on the SO and SSO micelles were quantitatively analyzed using a BCA protein assay kit. The safety and biocompatibility of the SSO micelles as drug carriers were confirmed by cytotoxicity and hemolysis tests. Additionally, DOX was loaded into the micelles as a fluorescent probe to test the long circulation effects of the SSO micelles. This work focuses on comparing the biocompatibility and long circulation properties of SO and

barrier created by strong hydrogen bonding of PEG ether groups with surrounding water molecules is able to resist proteins from approaching the surface of these nanoparticles.12 However, PEG coating has been reported to have several disadvantages in vivo applications, such as a lack of functional groups for postmodification,13,14 potential degradation in vivo,15−17 and most importantly accelerated blood clearance of nanoparticles after a second dose resulting from the generation of anti-PEG antibodies in the immune system. These disadvantages lead to fatal drawbacks in clinical applications.18,19 In recent years, Jiang and co-workers identified poly(zwitterionic) materials,20,21 a family of chargeneutral materials that possess both cationic and anionic groups at microenvironments, that were nontoxic, nonimmunogenic, and simple to synthesize. These materials have attracted tremendous attention due to their superior resistance to nonspecific protein adsorption, which overcomes all the challenges associated with PEG-coated nanoparticles. Zwitterionic materials have been widely investigated to construct nanoparticles with long circulation time.22 They have been shown to have excellent protein resistance properties (less than 0.3 ng·cm−2 of protein adsorption) on hydrogel surfaces or nanoparticles.23 Li and co-workers have reported that PCBgrafted nanoparticles demonstrated increased stability in vitro in both saline solution and human blood serum when compared with their PEGylated counterparts.24 Among the various types of nanoparticles, biodegradable polymeric micelles are one of the most promising drug delivery platforms due to their excellent biocompatibility and in vivo degradability.25−27 Starch, a major dietary source of carbohy4386


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according to literature procedures with slight modifications.37 Briefly, 20 mg of SB-ST-OC or ST-OC was dissolved in 4 mL of DMSO, and this polymer solution was slowly dropped into 20 mL of deionized water (DI water) with stirring over 3 h to form a stable micelle solution. Afterward, DMSO solvent was removed using a dialysis bag (molecular weight cutoff, 3500 g·mol−1) and dialyzed for 24 h. An amount of 10 mg of SB-ST sample was dissolved in 10 mL of DI water to obtain the SB-ST micelles. Then, this polymer solution was treated 30 times under a condition of activating every 2 s for a duration of 3 s using a probe-type ultrasonicator at room temperature. 2.3.2. Size and Morphological Examination. Micellar size distribution and zeta potentials were measured by a Nano Zetasizer Measurement (Nano ZS, Malven, UK). The concentration of sample solution used for test was 1 mg·mL−1. Morphological examinations were performed by TEM (JEM-2100F, JEOL, Japan). 2.3.3. Protein Resistance Test. The micelles of SB-ST-OC and STOC were mixed with a 10 wt % BSA solution and 10 wt % human serum solution, respectively, and the final micelle concentrations were maintained at 0.5 mg·mL−1.38,39 The stability test was carried out by measuring micelle size as a function of time. The quantitative analysis of protein adsorption by the starch derivative micelles was performed according to the literature.40,41 Briefly, the micelles were dispersed in BSA solution, keeping the final concentrations of micelles and proteins at 0.15 and 0.25 mg·mL−1, respectively. After incubation for different periods of time, the proteinadsorbed micelles were separated using a high-speed centrifuge, and the precipitate was then washed with phosphate buffer solution (PBS) to remove the unanchored protein. The adsorbed protein was detached by immersion in a sodium dodecyl sulfate (SDS) PBS solution under the condition of sonication for 30 min. The quantitative analysis of the adsorbed protein was obtained according to the standard calibration curve of BSA using a BCA protein analysis kit (n = 6). 2.3.4. Hemolytic Toxicity. The membrane disruption of red blood cells (RBCs) was employed to evaluation the hemolytic toxicity of starch derivative micelles using a RBC hemolysis assay.42,43 Healthy red blood cells (HRBCs) were prepared in PBS at 5% hematocrit according to our previously published procedures.44 Then, 0.1 mL of the diluted HRBC suspension was added to 0.9 mL of water and 0.9 mL of PBS, which served as the positive control and negative control, respectively. The micelle groups were prepared by adding 0.1 mL of the diluted HRBC suspension into 0.9 mL of SSO or SO micelle solution, and the micelle concentrations were from 50 to 400 μg·mL−1. Then, the micelle systems were incubated at 37 °C for 4 h to evaluate their hemolytic toxicity. At the end of the test, the micelle systems were centrifuged, and the photos of the centrifuge tubes were taken. Furthermore, the supernatant was measured to detect the hemoglobin leakage, and the percent hemolysis of the HRBCs with various samples was obtained based on the following equation

SSO micelles and offers a better drug delivery candidate for long circulation time in vivo.

2. MATERIALS AND METHODS 2.1. Materials. 1,3-Propane sultone was purchased from Jiangsu Mengde Electroplate Chemical Product Co., Ltd. (Jiangsu, China). 1Chloro-3-(dimethylamino)propyl hydrochloride (CDMAP·HCl) (98%) was purchased from Alfa Aesar Chemical Co., Ltd. (Tianjin, China). Starch (amylose, Mw 1.01 × 106) was purchased from Jiangtian Biotech (Tianjin, China) and dried under reduced pressure at 100 °C for 12 h before use. Octanoyl chloride was purchased from Aladdin Chemistry Co., Ltd. (Shanghai, China). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and Hoechst 33258 were purchased from Beyotime (Shanghai, China). Pyrene and doxorubicin hydrochloride (DOX·HCl) were purchased from Sangon Biotech Co., Ltd. (Shanghai, China). All other chemicals were of analytic grade and used as received. 2.2. Synthesis and Characterization of the Starch Derivatives. 2.2.1. Synthesis of the Starch Derivatives. Three different types of amphiphilic starch derivatives were synthesized and used to prepare different micelles as drug delivery carriers. 3-Dimethyl(chloropropyl) ammonium propanesulfonate (DCAPS) was chosen as a type of sulfobetaine (SB) to prepare the zwitterionic sulfobetaine-modified starch (SB-ST). The DCAPS and DCAPSmodified starch were synthesized according to previously reported methods from our lab.33 DCAPS was obtained by reacting 1,3-propane sultone with 1-chloro-3-(dimethylamino)propyl (CDMAP). SB-ST was synthesized using a Williams etherification reaction (Scheme 1A). Briefly, 2.78 g of starch was dispersed in 10 mL of water (containing 11 wt % of NaOH). Then, 10 mL of DCAPS solution was added to the starch solution, and the reaction was carried out with stirring at 60 °C for 6 h. Subsequently, the reaction system was poured into cold methanol (10 times the reaction system volume). The precipitate was recovered and washed with methanol 3 times. Eventually, the product was dried in a vacuum oven for 12 h to constant weight. The octanoyl grafting of SB-ST was carried out by esterification between the acyl chloride group and the −OH of SB-ST in the dimethylacetamide (DMAc) medium, and the final product was named SB-ST-OC.34 Briefly, SB-ST (1.0 g) was added to 20 mL of DMAc with stirring at 135 °C for 2 h under a nitrogen atmosphere. Then, 0.1 g of 4-dimethylaminopyridine (DMAP) was added to activate the −OH group of starch. Subsequently, 20 mL of DMAc containing different amounts of octanoyl chloride was added dropwise over 1 h at 100 °C, and the reaction was carried out for 6 h with stirring. The final reaction system was poured into cold ethanol. The product was washed with ethanol 3 times and dried in a vacuum oven to constant weight. The counterpart starch derivative, starch octanoate (ST-OC), only contained a hydrophobic segment without a zwitterionic group and was obtained by the same procedures where SB-ST was substituted with raw starch. 2.2.2. Characterization of SB-ST, SB-ST-OC, and ST-OC. 1H NMR spectra of the samples were recorded on a 400 MHz (AVANCE III, Bruker, Germany) spectrometer operated at room temperature using deuterated dimethyl sulfoxide (DMSO-d6) as a solvent and tetramethylsilane (TMS) as the internal standard. Fourier transform infrared (FT-IR) spectra of the samples were obtained using an IR spectrometer (WQF-510A, Rayleigh, China) with KBr as the substrate. The critical micelle concentrations (CMCs) of the synthesized SBST-OC and ST-OC with different graft ratios were determined using pyrene as a fluorescence probe.35,36 Briefly, a series of micelle solutions with different concentrations were prepared and introduced into prelabeled flasks containing pyrene. Then, the micelle systems were heated for 3 h at 65 °C and cooled overnight for an equilibration. The fluorescence spectra were recorded with the excitation wavelength of 338 nm using an F-4500 fluorescence spectrophotometer. Then, CMC values were calculated from the emission intensity ratios of I338 to I335 versus pyrene concentrations. 2.3. Preparation and Characterization of the Starch Derivative Micelles. 2.3.1. Preparation of the Starch Derivative Micelles. The micelles of SB-ST-OC or ST-OC were prepared

Percent hemolysis (%) =

(A sample − A negative) (A positve − A negative)

× 100 (1)

where Asample, Anegative, and Apositive represent the absorbances measured from the sample, PBS, and water groups, respectively (n = 6). To preclude the potential false positive result caused by endotoxin, all of the tests were carried out under aseptic conditions. The endotoxin levels in SSO and SO micelles were detected using an endotoxin assay kit (Jining, China) according to the manufacturer’s protocol. 2.4. Studies of DOX-Loaded Micelles. 2.4.1. Preparation of DOX-Loaded Micelles. Doxorubicin base (DOX) was obtained by reacting DOX·HCl with excess PBS (pH = 8.0) under stirring for 5 h in the absence of light. Afterward, the reaction mixture was centrifuged at 8000 rpm for 20 min to remove the residual drug compound that is not neutralized; the precipitate was then lyophilized for encapsulation. The DOX-incorporated micelles of SB-ST-OC and ST-OC were prepared as mentioned previously using the same procedure described in section 2.3.1 but with 1 mg of DOX added to the 4 mL fraction of DMSO. After dialysis against DI water, the unloaded DOX was 4387


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ACS Applied Materials & Interfaces removed by filtering the samples through a microporous membrane with 0.22 μm pore size. The loaded DOX in micelles was determined by measuring the absorbance at 488 nm with a fluorescence spectrophotometer. Drug loading (DL) and drug encapsulation efficiency (DE) were calculated using the following formulas

EE (%) =

mass of DOX encapsulated in micelles × 100% mass of feeding DOX

(2)

DL (%) =

mass of DOX encapsulated in micelles × 100% mass of DOX loaded polymer

(3)

A quantitative determination of cellular phagocytosis of micelles by flow cytometry was performed as follows. Briefly, RAW 264.7 cells were seeded in a 6-well plate (5 × 105 cells per well) and incubated with micelles as described above. Then, cells were harvested and suspended in 0.5 mL of PBS containing 1% PFA (paraformaldehyde) for fluorescence analysis by flow cytometry using a FACSAria Cell Sorter (Becton Dickinson BD). All data of the mean fluorescence signal were obtained from a population of 20 000 cells. Cells without micelle treatment and treated with DMEM alone were used as negative control groups. Cell-associated DOX was detected with the excitation wavelength and emission wavelength at 485 and 595 nm, respectively. 2.5.4. Cytokine Secretion and Cell Morphology Observation. The murine macrophage RAW 264.7 cell line was used to investigate the immune response of macrophage against the starch derivative micelles. RAW 264.7 cells were seeded in a 24-well plate at a density of 2 × 105 cells per well. After cultured for 24 h, the cells were stimulated with lipopolysaccharides (LPS: 1 μg·mL−1), PBS, and micelles. The cells treated with the PBS group served as a negative control which was set as 0% activation, and those treated with LPS served as positive control which was set as 100% activation. After the RAW 264.7 cells were stimulated for 24 h, cell morphologies were investigated using an optical microscope (IX71, Olympus, Japan) and scanning electronic microscopy (SEM, S-4800, Hitachi, Japan). Meanwhile, secretion of IL-6 and TNF-α from the cells was detected using mouse ELISA antibody pair sets (Invitrogen, Carlsbad, CA). The percentage activation was calculated based on the formula

2.4.2. In Vitro Release of DOX. Drug release profiles of DOX from SSO and SO micelles were investigated using a dialysis method. An amount of 1 mL of DOX-loaded SSO and SO micelle solutions was sealed in a dialysis bag and immersed into 30 mL of PBS, respectively.45,46The tests were performed at 37 °C under the condition of shaking horizontally at 100 rpm in an incubator shaker. At each time interval, 1 mL of dissolution medium was taken for detecting the DOX content and replaced with fresh PBS to prevent drug saturation. The released DOX from SSO and SO micelles was measured using a fluorescence spectrophotometer at a wavelength of 488 nm (n = 3). 2.5. In Vitro Cell Studies. 2.5.1. Cell Culture. Mouse macrophage RAW 264.7 cell line47 and HepG2 cell line48 were provided by Tianjin University of Traditional Chinese Medicine, and the culture methods were according to the literature. The cells were regularly monitored, and the medium was changed once in 3 days. 2.5.2. Cell Viability Assay. The cytotoxicity of the starch derivative micelles was analyzed using a methyl thiazolyl tetrazolium (MTT) viability assay. RAW 264.7 cells were seeded in a 96-well plate (approximately 104 cells per well) and grown in DMEM. After a 24 h incubation, the old culture medium was aspirated, and fresh DMEM containing the SSO/SO micelles (from 7.8 to 250 μg·mL−1) was added. The pure fresh medium group was set as a control. After further incubation for 48 h, 50 μL of MTT was added, and the cell culture plate was placed in an incubator for 4 h. The absorbance was measured using an ELISA reader (Multiskan MK3, Thermo Scientific, USA) at a wavelength of 490 nm. Cell viability was calculated according to eq 4. The viability of micelle-treated HepG2 cells was also investigated using the above method. Furthermore, the survival of HepG2 cells was investigated by acridine orange and propidium iodide (AO/PI) staining. At the end of the test, the HepG2 cells were stained by AO (0.67 μM) and PI (75 μM), respectively. After washing 3 times using PBS, a fluorescent microscopy was employed to observe the cell morphology.

Cell viability (%) =

A sample Acontrol

× 100

Percent activation (%) =

(Csample − Cnegative) (C positive − Cnegative)

× 100 (5)

where Csample, Cnegative, and Cpositive represent the values from the sample, PBS and LPS groups, respectively (n = 6). 2.6. Pharmacokinetics Study. Male Sprague−Dawley rats that weighed 200−220 g were obtained from Tianjin Shanchuanhong Laboratory Animal Technology Co., Ltd. (Tianjin, China). All of the experiment procedures were approved by the Animal Ethics Committee of Tianjin University of Traditional Chinese Medicine. Rats were randomly divided into three groups (each group contained six rats). The SD rats were intravenous administered DOX·HCl, DOXloaded SO micelle, or DOX-loaded SSO micelle at the dose of 2.5 mg· kg−1 body weight. At each time point (5, 15, 30, 60, 120, 240, 360, 480, 600, and 840 min), 0.4 mL of blood was collected from the retroorbital plexus of the mouse under anesthesia and was placed in a heparin-pretreated centrifuge tube. Then, the samples were centrifuged at 4000 rpm for 10 min to collect plasma which was stored in a refrigerator at −20 °C condition. The DOX concentration was determined according to the published literature using HPLC analysis with a fluorescence detector.39 2.7. Statistical Data Analysis. Significant differences were evaluated using Origin 8.0 (Graph Pad Inc., San Diego, CA). The results were reported as mean ± the standard deviation (SD). The differences between treatment groups were assessed using a student’s t-test (two-tailed). Pharmacokinetic parameters were obtained using the DAS 2.0 program (Drug and Statistics 2.0). All experiments were repeated at least three times. Difference was considered significant when the p-value was less than 0.05.

(4)

where Asample and Acontrol represent the absorbances of the sample and DMEM groups, respectively (n = 6). In addition to the blank micelles, DOX-loaded micelles were also evaluated by an MTT assay. The procedures were similar as above, and only the blank SSO and SO micelles were substituted with DOXloaded SSO/SO micelles (equivalent DOX concentrations: 0.31−15 μg·mL−1). 2.5.3. Cellular Phagocytosis Test. Cell phagocytosis of micelles carrying DOX (red fluorescence) and free DOX was evaluated by using confocal laser scanning microscopy (CLSM, LSM 710, Carl Zeiss, Germany) and flow cytometry (Becton Dickinson, Accrui C6, USA). For confocal microscope analysis, RAW 264.7 cells were seeded at 105 cells per well in a 24-well plate and incubated for 24 h. Then, old medium was aspirated, and fresh DMEM medium without red phenol and FBS was added. Cells were incubated with free DOX, DOX-loaded SSO, or SO micelles for another 1 and 4 h at a concentration of 15 μg·mL−1. Additionally, Hoechst 33258 was used to stain the cell nuclei, and microscope images were observed with a CLSM.37,49

3. RESULTS AND DISCUSSION 3.1. Synthesis and Characterization of the Starch Derivatives. In this work, we present a rationally designed starch derivative with superhydrophilic zwitterionic SB and hydrophobic octane functional groups (Scheme 1A) based on the biodegradability and excellent biocompatibility of raw starch. The starch derivatives of SB-ST-OC were synthesized in two steps. First, 3-dimethyl(chloropropyl) ammonium propanesulfonate (DCAPS), a type of sulfobetaine (SB), was introduced as a zwitterionic group to the starch backbone by an 4388


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chemical shift at 5.1 ppm. The graft ratio of OC or SB, defined as the number of OC or SB per AGU, was calculated using the proton peak areas of −CH3 of OC (at 0.9 ppm, peak 7), N(CH3)2 of SB (at 3.0 ppm, peak d), and the proton peak areas of starch (at 5.1 ppm, peak 1) in spectrum. The graft ratio of OC increased with the feeding amount of OC at fixed SB content, as shown in Table 1. The FT-IR spectra of raw starch (A), SB-ST (B), and SB-STOC (C) are shown in Figure 2. Compared with raw starch, the

etherification reaction. Then, the hydrophobic side chains were combined by esterification reactions of the hydroxyl group (−OH) on the starch main chain and octanoyl chloride, as shown in Route 1. The target amphiphilic SB-ST-OC was used to prepare the SSO micelles. The introduction of hydrophobic groups can provide the hydrophobic force to form micelles, and the hydrophobic region can also provide sufficient space to load a hydrophobic drug. As we know, the interaction of nanoparticles with opsonin is a critical process and the first step in nanoparticle recognition by the immune system once the nanoparticles enter the bloodstream. Therefore, the ability of micelles to remain stable in blood and resist blood protein adsorption plays a vital role in long-circulation drug delivery. For this purpose, the hydrophilic zwitterionic SB with superprotein resistance was introduced on the surface of the micelles to endow them with excellent protein resistance, avoid recognition and phagocytosis by macrophages, and ultimately achieve the purpose of longer circulation time in the bloodstream. As a control, the SO micellar polymer (ST-OC) only containing the hydrophobic segment without zwitterionic group was included (Route 2). Scheme 1B illustrates the starch derivatives’ self-assembly into SO and SSO micelles. The representative structures of raw starch (A), ST-OC (B), and SB-ST-OC (C) were characterized by 1H NMR. As shown in Figure 1, the methylidyne proton of CH−O (number 2, 3, 4,

Figure 2. FT-IR spectra of (A) raw starch, (B) SB-ST, and (C) SB-STOC.

FT-IR spectrum of SB-ST showed characteristic absorption bands at 1483 and 1201 cm−1 resulting from the N−CH3 stretching vibrations and SO asymmetric vibrations of the zwitterionic SB groups. The FT-IR spectrum of the SB-ST-OC sample showed the characteristic peaks of the SB-ST sample and new strong adsorption bands at 2925, 2850, and 1750 cm−1 that corresponded to −CH3, −CH2−, and CO groups, respectively. On the basis of the 1H NMR and FT-IR spectral analyses, we confirmed that the targeted starch derivatives were successfully synthesized. As we known, CMC is a typical characteristic for amphiphilic polymers that indicates micelle formation and thermodynamic stability, and the self-assembly behaviors of ST-OC and SB-STOC with different grafting ratios were investigated by fluorometry. The fluorescence intensity versus polymer concentrations were plotted in Figure 3B. There is an abrupt change of I338/I335 value as the polymer concentration increased which was attributed to the polymer self-assembly into micelles in water. Meanwhile, pyrene was transferred into the micelles due to their high sensitivity to hydrophobic environment. The

Figure 1. 1H NMR spectra of (A) raw starch, (B) ST-OC, and (C) SB-ST-OC in DMSO-d6.

5) and methylene proton of CH2−O (number 6) in the anhydroglucose (AGU) have a chemical shift at 3.4−4.0 ppm. The methylidyne proton of O−CH-O (number 1) has a

Table 1. Physicochemical Properties of Synthesized Starch Derivatives: SB-ST, ST-OC, and SB-ST-OCf feeding ratioa samples SB-ST ST-OC SB-ST-OC

ST/SB-ST:OC e

---1:1 1:4 1:3 1:7

graft ratiob SB 0.17 ----e ----e 0.17 0.17

CMC (mg·L−1)

OC e

e

---0.10 1.91 0.11 1.81

---84.71 ± 3.29 ± 94.11 ± 3.82 ±

4.50 0.28 5.91 0.35

Dnc (nm) 49.40 122.25 61.31 138.72 73.85

± ± ± ± ±

6.15 1.30 0.82 3.26 0.23

PId 0.25 0.15 0.12 0.15 0.15

± ± ± ± ±

0.02 0.03 0.01 0.04 0.04

a

The feeding ratio represents the molar feeding amount of octanoyl chloride corresponding to per AGU. bThe graft ratio of OC or SB was defined as the number of OC or SB per AGU. cDn represents the hydrodynamic diameter of the micelles. dPI represents the polydispersity index of the micelles. e“----” means that the item does not exist or could not be measured for the sample. fDn and PI represent the mean ± standard deviation (n = 3). 4389


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Figure 3. Characterization of the SO micelle and SSO micelle. (A) Size distribution of SO and SSO micelles. (B) Variation of intensity ratio (I338/ I335) versus logarithm of SO micelle and SSO micelle concentrations. (C) TEM image of SSO micelles. (D) TEM image of SO micelles.

Figure 4. Protein resistance of SO and SSO micelles. Size and zeta potential change of SO and SSO micelles in different media at 37 °C as a function of time. (A) In 10% BSA solution. (B) In 10% human serum solution. (C) BSA adsorption by SO and SSO micelles after 4 and 8 h of incubation at 37 °C. Asterisks (*) denote statistical significance of SSO micelles vs SO micelles (*p < 0.05).

CMC results demonstrated that an increase of OC grafting ratio resulted in a decreased CMC, indicating that these

polymers formed more thermodynamically stable micelles under highly diluted conditions. SB0.17-ST-OC1.81 provides a 4390


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ACS Applied Materials & Interfaces lower CMC (3.82 mg·L−1) than its lower OC grafting ratio counterpart SB0.17-ST-OC0.11 (94.11 mg·L−1). A similar trend was also exhibited by ST-OC1.91 and ST-OC0.10, which had CMCs of 3.29 and 84.71 mg·L−1, respectively. However, the CMC of the SB-ST group could not be obtained using the same method due to its different micelle formation mechanism. Pyrene is highly sensitive to polarity of the environment. However, the “core” of the SB-ST micelle is driven by hydrogen-bonding interactions rather than hydrophobic forces, resulting in the failure to transfer pyrene into the core of the SB-ST micelle and a measurement failure. Groups of starch derivatives with the same SB grafting ratio (DSSB = 0.17) and higher OC grafting ratio (DSOC = 1.91 for ST-OC and DSOC = 1.81 for SB-ST-OC) were used for the further studies in vitro and in vivo due to the stability of the corresponding SB-ST, ST-OC, and SB-ST-OC micelles. The micelles prepared from polymer ST-OC were called “SO micelles”. To prove the critical role of SB modification, which is important in protein resistance and beneficial for increasing resident times in the bloodstream, the counterpart micelle was prepared from SB-ST-OC. These micelles were decorated with zwitterionic SB on the surface of the SO micelles and were called “SSO micelles” (Scheme 1). 3.2. Formation and Size Distribution of Starch Derivative Micelles. In addition to thermodynamic stability, hydrodynamic size is another important factor for nanoparticle applications. It is generally accepted that micelles can be cleared rapidly from blood when the size was larger than 150 nm, resulting in poor circulation time in vivo. It was reported that micelles smaller than 5.5 nm would be removed by the kidneys, which would also decrease circulation time of micelles in vivo.50 Fortunately, the sizes of the prepared SB-ST-OC and ST-OC micelles ranged from 60 to 140 nm, falling into the suitable nanosize range for use as long-circulating drug carriers (Table 1). Meanwhile, the hydrodynamic diameters of the SB-ST-OC and ST-OC micelles reduced with increasing graft ratios of OC, where each of the groups exhibited a narrower distribution. The successful formation of starch derivative micelles was also confirmed by TEM (Figure 3C and D). The micelles exhibited spherical morphologies with average diameters of approximately 50 nm. On the other hand, the micellar sizes detected by TEM were smaller than those obtained from Nanosizer measurements, which was attributed to the micelles shrinking during the drying process of the TEM sample. 3.3. Protein Resistance of Starch Derivative Micelles. In addition to the suitable size and good thermodynamic stability, an additional enormous challenge for the intravenous administration of micelles is the effect of various proteins that tend to adhere to the surface of micelles. These proteins label the micelles as foreign bodies and are quickly cleared by the MPS before the micelle can reach its target site. Therefore, enhancing the protein resistance of micelles is of great importance in achieving the long-term delivery. Herein, the protein resistance properties of the micelles in complex medium was investigated by measuring the size changes of micelles in 10% BSA and 10% human serum solutions at 37 °C. As shown in Figure 4A, the size of the SO micelles increased by approximately 15 nm in a very short period of time and reached 125 nm (from 61 nm) by the end of the 11-day test period, which indicated that significant protein adsorption led to aggregation of the micelles. The same trend was also observed when the micelles were placed in a 10% human serum solution (Figure 4B), and the micelle sizes reached 130 nm

finally (from 61 nm). However, the SSO micelles with zwitterionic modifications were stable in 10% BSA and 10% human serum solutions without obvious size changes at the end of 11-day incubation; thus, the SSO micelles demonstrated excellent protein resistance properties. Comparing the two types of micelles, the SSO micelle showed slightly negative zeta potential, approximately −2.4 ± 0.2 mV, while the SO micelle was negatively charged (−24.9 ± 1.2 mV). Under normal circumstances, the electrostatic repulsive force among the micelles can make the system under a more stable state. Actually, the stability based on electrostatic repulsion can be kept only in simple environments. In a complex physiological environment, a lot of protein adhesion would destroy this balance as shown in Figure 4. Although the zeta potential of SSO micelle was approximately equal to zero (−2.4 ± 0.2 mV), the hydrophilic SB groups could form a tight hydration layer by electrostatic interactions. This layer could prevent the contact of micelle with protein and provide protection for micelles.51 Furthermore, in BSA solution, the zeta potentials increased from −24.9 to −16.5 mV as the micelle size increased from 61 to 125 nm, indicating the protein adsorbed onto the micelles. The same phenomenon for SO micelle was also observed in human serum solution. This phenomenon could be explained by that the zwitterionic-modified SSO micelles contain equal amounts of quaternary ammonium cations and sulfonate anion groups on the micelle surface. At a microscopic environment, a strong hydration layer via electrostatic interactions was formed around the SSO micelles. However, the hydrogen bonding interactions that form the SO micellar hydration layer are weaker than electrostatic interactions. As a result, the SO micelles have a weaker protein resistance than SSO micelles, resulting in protein adsorption and size increases. These results imply that SSO micelles are not prone to aggregation in a physiological environment and are more suitable as drug carriers in complex media such as bloodstreams. On the basis of the evaluation for the micelle stability in BSA solution, we further quantitatively analyzed the amount of protein adsorption by the SO and SSO micelles. The equilibrium amounts of BSA adsorbed on SSO micelles was less than that of SO micelles after 4 h of incubation (Figure 4C). As the time increased to 8 h of incubation, the SSO micelles still showed minimal protein adsorption; however, the protein adsorption of the SO micelles increased significantly. This quantitative analysis further demonstrates the excellent protein resistance properties of SSO micelles. These results imply that SSO micelles could potentially be used for in vivo drug delivery. 3.4. Hemolysis and Toxicity Analysis. A serious limitation of nanocarrier in vivo application is their nonspecific interactions with blood proteins, which could result in hemolysis. Thus, a basic test was necessary to understand the interaction of nanoparticles with HRBCs. The formation of a hydration layer around polymer surfaces of hemocompatible polymers has been recognized as an essential characteristic to maintain their protein resistance properties.52 It was expected that the starch derivative of SB-ST-OC would be hemocompatible due to its superhydrophilicity and excellent protein resistance properties. To study the hemolytic potential of the SO and SSO micelles, HRBCs were treated with 0−400 μg·mL−1 of SO and SSO micelles over 4 h at 37 °C. As shown in Figure 5, compared with the positive control (DI water), which registers 4391


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the hydroxyl groups of the ST-OC micelles, which led to stronger water shield layers that prevented the contact of SBST-OC micelles with blood cell membranes. In addition to hemolysis, RAW 264.7 cells and HepG2 cells were chosen as in vitro models to assess the cytotoxicity of the micelles. Figure 6A and B shows the viabilities of RAW 264.7 cells and HepG2 cells cultured in DMEM for 48 h containing SO and SSO micelles at various concentrations. Both types of micelles were highly cytocompatible, even at concentrations up to 250 μg·mL−1, and this could be caused by the essential of starch, as a representative of polysaccharides, with good biocompatibility. Furthermore, the AO/PI staining images (Figure 6C) showed that only several dead cells (red) can be observed at all of the sample groups, indicating the good cytocompatibity of the micelles. These results suggest that the SSO micelles are a promising drug carrier owing to their excellent hemocompatibility, cytocompatibility, and protein resistance properties. 3.5. Macrophage Responses to Starch Derivative Micelles. 3.5.1. Cellular Phagocytosis Study. In the phagocytosis test, DOX, a fluorescent probe, was incorporated into the micelles. The cellular phagocytosis of DOX-loaded SO and SSO micelles against RAW 264.7 cells was analyzed using CLSM and flow cytometry. The drug loadings in SSO and SO micelles were accurately calculated to be 5.38% and 6.77% (Table S1), respectively. The DOX-loaded SSO and SO micelles exhibited similar characteristics in the cell viability tests against HepG2 cells (Figure S1). Additionally, the release profiles of the micelle groups were investigated in PBS buffer at pH 7.4. The DOX release curves of micelle groups were plotted in Figure S2, and no obvious difference of the two types of micelles in drug release behavior was found. The results shown in Figure 7 are the cellular images after the RAW 264.7 cells were incubated with different samples for 1

Figure 5. Percent hemolysis of HRBCs incubated with starch derivative micelle systems of varying concentrations. Inset photograph: hemolysis assay of SO/SSO micelle-treated HRBCs compared to the DI water and PBS groups. P: positive; N: negative; Asterisks (*) denote statistical significance of SSO micelle vs SO micelle (*p < 0.05).

≈100% hemolysis, micelle-treated HRBCs showed a hemolysis of less than 2% up to 200 μg·mL−1, indicating their excellent blood compatibility. This was confirmed by the optical photograph (Figure 5) of samples, in which the DI water group showed significant red color in the supernatant, while both micelle groups showed clear supernatant at a 400 μg·mL−1 treatment concentration and undamaged HRBCs at the bottom of the tube. It is interesting that the zwitterionic-modified micelle group exhibited better antihemolytic activity, even at a higher concentration of up to 400 μg·mL−1, compared to the SO micelle group, which showed a hemolysis percentage of up to 5.26% (the hemolysis of SSO micelle was 1.88%). This was attributed to the stronger hydration capability of the zwitterionic moieties of the SB-ST-OC micelles compared to

Figure 6. In vitro cytotoxicity of SSO and SO micelles against RAW 264.7 cells (A) and HepG2 cells (B) after 48 h incubation with pure DMEM as a control. (C) AO/PI staining images of HepG2 cells cultured with different concentrations of SSO and SO micelles. 4392


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Figure 7. Representative CLSM images of RAW 264.7 cells treated with free DOX, DOX-loaded SO, and SSO micelles after incubation for 1 and 4 h. A bar is 34 μm.

and 4 h. The cellular phagocytosis of the two types of micelles was significantly less than that of free DOX. As shown in CLSM images, DOX intracellular distributions in the micelle groups were quite different from the free DOX group. After incubation in free DOX solution for 1 h, the fluorescence was mainly distributed in the cell nuclei, which is attributed to the rapid diffusion mechanism of small molecules. However, the red fluorescence intensities near nuclei in the DOX-loaded micelle groups were significantly weaker compared to free DOX group. Comparing the three test groups, the DOX fluorescence of the free DOX group was mainly located in nuclei. However, SSO and SO micelle groups were detected in both nuclei and cytosol, and the majority of DOX fluorescence was located in cytosol. The red fluorescence of DOX was significantly weaker in the SSO micelle group compared to the SO micelle group. As time extended to 4 h, the same tendency was observed in the three sample groups, and the SSO micelle group with zwitterionic modification showed the weakest fluorescence intensity.

The phagocytosis of the DOX-loaded micelles was further quantified via flow cytometry. After each period of incubation for 1 or 4 h, the fluorescence intensities of RAW 264.7 cells treated by different sample groups were in the sequence free DOX > SO micelle > SSO micelle (Figure 8). This trend was in agreement with the results of CLSM (Figure 7). The fluorescence intensity of free DOX was 1.5- and 3.7-fold to DOX-loaded SO micelles and SSO micelles at 1 h, respectively. This result was supported by the literature that the faster diffusion mechanism of free DOX and the quench effect of the DOX were in the core of micelles.53,54 Moreover, comparing the two types of DOX-loaded micelles, the fluorescence intensity in the DOX-loaded SSO micelle group was significantly weaker than that in the SO micelle at both time points. This phenomenon could be attributed to zwitterionic moieties that prevented the contact of micelles to cell membranes, thereby leading to less phagocytosis of SSO micelles compared to the SO micelles. These results were in agreement with the protein resistance and hemolysis tests. 4393


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Figure 8. (A) Flow cytometry analysis of cellular phagocytosis against RAW 264.7 cells treated by free DOX, DOX-loaded SO, or SSO micelles after 1 and 4 h incubation. (B) and (C) Quantitive analyses of the mean fluorescence intensity for 1 and 4 h, respectively.

3.5.2. Effect of Micelles on Cytokine Secretion and Cell Morphology. In addition to the visualized fluorescence pictures, the effects of micelles on macrophages were evaluated by cytokine secretion and cell morphology observations. During the macrophage stimulate−response process, activated macrophages secrete soluble cytokines which can recruit more macrophages to remove the foreign substances in the body. Thus, we examined the secretions of TNF-α and IL-6 from RAW 264.7 cells. To explain the effect of micelles on macrophages more directly, the ELISA results were converted into activation rates (percentage activation) according to the formulation (eq 4) in section 2.5.4. As shown in Figure 9A and B, there were relatively low releases of TNF-α and IL-6 by both the SO and SSO micelle groups, and they were increased in all groups in a dose-dependent manner. Compared to the SSO micelle group at a concentration of 100 μg·mL−1, the SO micelle group induced significantly higher secretion levels of IL-6 and TNF-α. Compared with the positive control (LPS) which registers ≈100% activation, micelle-group-treated cells showed a little activation of less than 5% up to 50 μg·mL−1 (both IL-6 and TNF-α). However, the SO micelle group showed a significantly higher activation rate at a higher micelle concentration up to 100 μg·mL−1 compared to the SSO micelle group (both IL-6 and TNF-α). This means that the SO micelles had a relative

higher potential to trigger the activation of macrophages and have been cleared by macrophages, compared to SSO micelles. There is a concern that higher levels of IL-6 and TNF-α can also be obtained when nanomaterials are contaminated with endotoxins. To address these issues, the micelle samples were tested for endotoxin levels, and the results show that 1 mg· mL−1 of SSO and SO micelle solutions produced endotoxin levels of 0.025 and 0.023 EU·mL−1. The results were lower than the upper detection limit (0.5 EU·mL−1, FDA, US) and could preclude false positives caused by endotoxins. In addition to the cytokine secretion, light microscope and SEM images depicting cell morphology were also obtained. In the control group (Figure 9C and G), the cells in standard culture conditions exhibited a round-shaped morphology, which was the normal state. In sharp contrast to the control group, cell morphologies changed dramatically upon LPS stimulation (Figure 9F and J), and many cells exhibited spread morphologies, which represented the activated state. Compared with the control group, the SO micelle (Figure 9D and H) and SSO micelle (Figure 9E and I) groups produced cells that were almost round in shape, with fewer cells exhibiting deformed morphologies. Moreover, more cells presented spread states in the SO micelle group than in the SSO micelle group, indicating that the SSO micelles were less capable of triggering the activation of macrophages. 4394


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Figure 9. Effects of SSO and SO micelles on macrophages. ELISA analysis of IL-6 (A) and TNF-α (B) expressed as percentages in SO and SSO micelle-treated RAW 264.7 cells. The amounts of cytokines secreted in negative control (PBS) and positive control (LPS) groups were taken as 0 and 100%, respectively. Light microscopy (original magnification 100×) and SEM (original magnification 400×) images of RAW 264.7 cells after 24 h treatment with DMEM (C, G), SO micelle (D, H), SSO micelle (E, I), and LPS (F, J), respectively. Asterisks (*) denote statistical significance of SSO micelle vs SO micelle (*p < 0.05).

3.6. In Vivo Circulation Stability of Drug-Loaded Starch Derivative Micelles. To further confirm the longcirculation properties, the pharmacokinetic behaviors of free DOX and both DOX-loaded micelles in rats were investigated, and the plasma concentration−time curves were plotted in Figure 10. The free DOX experienced rapid elimination from the bloodstream and could not be detected after 2 h of administration. In contrast, both of the micelle groups demonstrated extended circulation time. The concentration of

DOX in SSO micelles was higher than that of the SO micelle group at any time point after administration. Table 2 summarizes the main pharmacokinetic parameters obtained from DAS software. The results demonstrate that the Table 2. Pharmacokinetic Parameters of DOX for Intravenously Administrated Free DOX, DOX-Loaded SO Micelles, and DOX-Loaded SSO Micelles (n = 6)a parameter

free-DOX

SO micelle/DOX

SSO micelle/DOX

t1/2β (min) AUC0‑∞ (μg/L*min) MRT0‑∞ (min)

12.02 ± 8.67 88.06 ± 24.97

101.87 ± 60.17 600.54 ± 89.86

198.38 ± 22.84* 937.99 ± 482.11*

11.42 ± 5.36

76.92 ± 14.77

121.52 ± 16.96*

a

Asterisks (*) denote statistical significance of SSO micelles vs SO micelles (*p < 0.05).

DOX-loaded SSO micelle bearing zwitterionic SB groups showed significantly increased half-lives (16.05- and 1.95-fold greater than those of free DOX and SO micelles, respectively) and mean residence times (MRTs, increased by 6.73- and 1.58fold compared to free DOX and SO micelles, respectively). Additionally, the increase of AUC0−∞ could further confirm the extended circulation time of DOX-loaded SSO micelles in vivo (increased by 11.29- and 1.56-fold compared to free DOX and SO micelles, respectively), which was consistent with the previous in vitro results. It is well-known that the surface properties of nanoparticles are the main factors that affect blood clearance. The SSO micelles with SB groups on the surface of the micelles reduced plasma adsorption, suppressed

Figure 10. Plasma DOX concentration−time curve for intravenously administrated free DOX, DOX-loaded SO micelles, and DOX-loaded SSO micelles. 4395


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Scheme 2. Schematic Illustration of Protein Resistance of SO/SSO Micelles and Macrophage Behavior in Response to SO/SSO Micelles and the Long Circulation Mechanism of SSO Micelles

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the recognition and phagocytosis of micelles by macrophages, and eventually prolonged circulation time in blood. These in vitro results indicate that SSO micelles have better protein resistance and lower macrophage activation rates than SO micelles, which confirmed that the SSO micelles possessed the “stealth” properties and are promising in vivo carriers. Scheme 2 showed the different fates of the micelles upon entering bloodstreams. The SO micelles were susceptible to protein adsorption, which led to recognition by macrophages and, eventually, failure in drug delivery. In sharp contrast, the SSO micelles resisted protein adsorption, escaped from the recognition by macrophages, and possessed the potential for longer circulation time in blood. The in vivo results indicate that the SSO micelle can significantly extend the circulation time of the DOX, compared to the micelle without zwitterionic modification. The in vitro and in vivo results confirmed that the SSO micelles of SB-ST-OC possess “stealth” properties and are a promising drug delivery carrier for in vivo application.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.5b10811. Figure S1, toxicity of DOX-loaded SSO and SO micelles against HepG2 cells. Table S1, characteristics of DOXloaded SSO and SO micelles. Figure S2, in vitro drug release profiles of DOX-loaded SSO and SO micelles (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*Tel.: +86-22-27402893. Fax: +86-22-27403389. E-mail: [email protected] (Fanglian Yao). *Tel.: +86-22-59596170. Fax: +86-22-59596170. E-mail: [email protected] (Yuanlu Cui). Notes

The authors declare no competing financial interest.

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4. CONCLUSION

REFERENCES

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In this work, stealth nanoparticle platform utilizing micelles, based on superhydrophilic zwitterionic and hydrophobic octane binary graft modifications of starch, were developed as a novel drug delivery vector. In addition to the conventional examination of biocompatibility and potential medical applications by cell viability assays, this work has evaluated the nanomaterials from a perspective of macrophage response by detecting the secretions of IL-6 and TNF-α upon the macrophage phagocytosis of micelles. Compared with SO micelles, the zwitterionic-modified SSO micelles showed better protein resistance, better hemocompatibility, less cytotoxicity, and less macrophage activation potential in vitro. The pharmacokinetics results indicated that the DOX-loaded SSO micelles could prolong circulation time and delay the clearance of DOX better than SO micelles or free DOX. On the basis of these results, it can be concluded that the novel starch derivative of SB-ST-OC has excellent protein resistance, biocompatibility, and less potential to trigger the activation of macrophages and is an attractive candidate as a long-circulation vector in vivo. 4396


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