Biodegradable Zwitterionic Nanogels with Long Circulation for

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Biological and Medical Applications of Materials and Interfaces

Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery Yongzhi Men, Shaojun Peng, Peng Yang, Qin Jiang, Yanhui Zhang, Bin Shen, Pin Dong, Zhiqing Pang, and Wuli Yang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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ACS Applied Materials & Interfaces

Biodegradable Zwitterionic Nanogels with Long Circulation for Antitumor Drug Delivery Yongzhi Men,† Shaojun Peng,‡ Peng Yang,‡ Qin Jiang,‡ Yanhui Zhang,† Bin Shen,† Pin Dong,*,† Zhiqing Pang,*,§iD Wuli Yang,*,‡iD

†

Shanghai General Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200080, PR China ‡

State Key Laboratory of Molecular Engineering of Polymers & Department of Macromolecular Science, Fudan University, Shanghai, 200433, PR China

§

Key Laboratory of Smart Drug Delivery, Ministry of Education, Department of

Pharmaceutics, School of Pharmacy, Fudan University, Shanghai, 201203, PR China

KEYWORDS: zwitterionic nanogel, biodegradable, long circulation, drug delivery, reflux precipitation polymerization, reduction-responsive

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ABSTRACT: Zwitterionic nanocarriers have emerged as a new class of biocompatible nanomaterials with outstanding stealth capability in blood circulation. In this work, a novel biodegradable zwitterionic nanogels based on poly(sulfobetaine methacrylate) (PSBMA) was developed for reduction-responsive drug delivery to tumors. PSBMA nanogels were facilely fabricated by one-step reflux precipitation polymerization with the advantage of surfactantfree and time-saving. The disulfide bond not only endowed the nanogels degradability in reduction environment but also be modified with fluorescent group after partially reduction. In vitro release experiment disclosed that doxorubicin (DOX)-loaded PSBMA nanogels could hold the drugs firmly in physiological conditions (only 7% release in 24 h) and release the drugs rapidly and sufficiently in 10 mM glutathione (85% in 8 h). More interestingly, PSBMA nanogels displayed long circulation in blood after intravenous injection, and small change was found in half-life of nanogels between the first (34.1 h) and the second injection (30.5 h), indicating that there was no accelerated blood clearance phenomenon for these nanogels. Meanwhile, no obvious immunogenic response was detected after PSBMA nanogels were injected into BALB/c mice. Furthermore, PSBMA nanogels showed a high accumulation of 9.5% and 10.7% of injected dose per gram of tissue in tumors at 24 h and 48 h post intravenous injection, respectively. With outstanding long circulation time, high tumor accumulation, and sufficient drug release in reduction environment, DOX-loaded PSBMA nanogels demonstrated the strongest tumor growth inhibition effect among all the treatment groups in human hypopharyngeal carcinoma-bearing mouse models. Therefore, our study provided a facile drug delivery platform based on biodegradable zwitterionic nanogels and may have great potential in tumor drug delivery.

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1. INTRODUCTION The nanoparticle drug delivery systems have demonstrated enhanced therapeutic efficacy and lower toxicity of chemotherapeutics in tumor therapy, due to the enhanced permeability and retention (EPR) effect of nanoparticles in tumors.1-3 Among multitudinous nanoparticle drug delivery systems, nanogels have attracted much attention for their nanoscaled size, superior colloid stability, and flexibility in controlling the release of drugs.4-6 Therefore, numerous monomers, such as N-isopropyl acrylamide, vinyl caprolactam, oligo(ethylene glycol) methacrylate, methacrylic acid, and so on, have been applied to fabricate smart nanogels with temperature or pH-responsive property.7-10 Among nanogels fabricated by these monomers, poly(oligo(ethylene glycol) methacrylate) (POEGMA) nanogels can not only offer excellent temperature-responsive characteristics but also have a long circulation life in blood because of the polyethylene glycol (PEG) unit in POEGMA nanogels.11 However, recent research revealed that PEGylated nanoparticles often lose their long-circulating property and compromise tumor drug delivery when they are repeatedly administered in the same animal because of an accelerated blood clearance (ABC) phenomenon resulting from the generation of anti-PEG immunoglobulin antibodies.12 Therefore, developing new nanogels with stable long-circulation characteristic and low immunogenic response is still urgently needed for tumor drug delivery. Recently, several nanoparticles such as natural cell membrane-coated nanoparticles and zwitterionic polymer nanoparticles have aroused extensive attention on account of their better circulation time and negligible immunogenic response compared with PEGylated nanoparticles.13, 14 As an alternative to PEG, zwitterionic polymers possess both cationic and anionic groups but still remain charge neutrality as a whole. Due to the unique structure of zwitterionic polymers, zwitterionic nanoparticles have stronger water-binding capability than PEGylated nanoparticles as they are hydrated through strong electrostatic interactions while 3



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PEGylated nanoparticles interact with water molecules through hydrogen bonding.15 Therefore, zwitterionic nanoparticles can efficiently overcome the current disadvantages of PEGylated nanoparticles.16 Since pioneering work was done by Jiang et al. and Ishihara et al., zwitterionic polymers have been widely used in the application of stealthy nanoparticles.17,

18

Tong et al. used

zwitterionic phosphorylcholine polymer as hydrophilic segment to achieve the glutathione activatable photodynamic theranostics.19 Sun et al. applied zwitterionic sulfobetaine polymer to conjugate with polylactide and the obtained nanoparticles showed well-suppressed nonspecific interaction with biomolecules.20 However, these nanoparticles were mainly focused on micelles while zwitterionic nanogels were rarely reported, which might be ascribed to the lack of efficient and convenient methods to fabricate zwitterionic nanogels.21-23 In general, reverse emulsion polymerization was the most common method to prepare zwitterionic nanogels, due to the super hydrophilicity character of zwitterionic monomers. Jiang et al. firstly reported the synthesis of zwitterionic polymer nanogels based on carboxybetaine methacrylate (CBMA) by inverse emulsion polymerization.21 The obtained nanogels exhibited outstanding biocompatibility and excellent stability in fetal bovine serum. Liu et al. also developed a kind of zwitterionic multifunctional nanogel by copolymerizing ornithine methacrylamide with cross-linkable ﬂuorescent carbon dots by inverse emulsion polymerization.22 Although stable and biocompatible nanogels were elaborately prepared by inverse emulsion polymerization, large amounts of surfactants must be used in the preparation process as emulsifiers, thereby limiting their further application in biomedical application. Recently, Paulusse’ group presented stable zwitterionic poly(amido amine) nanogels by surfactant-free, inverse nanoprecipitation method.23 The fabricated nanogels displayed a clear zwitterionic swelling proﬁle in response to pH changes and exhibited high colloidal stability in fetal bovine serum medium. However, the inverse nanoprecipitation method process was divided into two steps and consumed several days in the whole fabrication process. Therefore, 4


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developing clean and convenient method to fabricate zwitterionic nanogels is still in urgent demand. In addition, the fast and sufficient drug release from nanogels is very important to kill tumor cells efficiently. As is known to us, the glutathione (GSH) concentration showed remarkable differences between in tumor cells (~10 mM) and in blood (~10 µM).24,

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Therefore, disulfide bond was widely applied in nanoparticles to respond to high concentration of GSH in tumor cells, thereby achieving reduction-responsive drug release of nanoparticles.26, 27 In this work, novel reduction-responsive zwitterionic nanogels as tumor drug delivery platform was facilely fabricated through by one-step reflux precipitation polymerization without adding any surfactants (Scheme 1). Sulfobetaine methacrylate (SBMA), which is commercial available and cheap, was selected as the zwitterionic monomer in this work. In fact, not only SBMA, other kinds of zwitterionic monomers can also be prepared by this method which provided a universal platform to fabricate zwitterionic biodegradable nanogels. The stability, biodegradability, cellular uptake, blood circulation, immunogenic response, and accumulation in tumor tissues of these nanogels were investigated. Finally, the antitumor efficacy of doxorubicin-loaded nanogels and the safety of nanogels were evaluated, which may pave the way for the wide application of PSBAM-based nanomaterials.

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Scheme 1. Schematic illustration of biodegradable zwitterionic PSBMA nanogels loaded with doxorubicin for antitumor therapy.

2. MATERIALS AND METHODS 2.1.

Materials

and

Instruments.

Sulfobetaine

methacrylate

(SBMA),

N,N′-

methylenebisacrylamide (MBA), and N,N’-bis(acryloyl) cystamine (BAC) were obtained from Alfa Aesar (UK). Glutathione (GSH), tris(2-carboxyethyl)phosphine (TCEP), 3-(4, 5Dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium

bromide

(MTT),

and

2,

2-

azobisisobutyronitrile (AIBN) were purchased from Sigma-Aldrich (USA). The details of chemical reagents used for POEGMA nanogels synthesis including were presented in our previous work.28 Doxorubicin (DOX) was purchased from Beijing Huafeng United Technology Company (China). Acetonitrile was purchased from Shanghai Lingfeng Company (China). Sulfo-Cyanine7.5 maleimide (Cy7.5 maleimide) was ordered from Lumiprobe (USA). Mouse IgM Enzyme-linked immunosorbent assay (ELISA) Kit and the Mouse IgG ELISA Kit were purchased from Multi Science (Hangzhou, China). Terminal deoxynucleotidyltransferase-mediated UTP end labeling (TUNEL) assay was purchased from Roche (Mannheim, Germany). Cell culture medium minimum essential media (MEM), fetal bovine serum (FBS) were purchased from Gibco (USA). All the chemicals and biological reagents were used as received unless otherwise mentioned. Human hypopharyngeal carcinoma cell line (FaDu) was obtained from ATCC (USA). Male BALB/c mice and nude mice aged 5-6 weeks were obtained from Shanghai Slac Laboratory Animal Co. Ltd (Shanghai, China). All animal experiment protocols were carried out according to the policies and regulations formulated by the Experimental Animal Ethics Committee of Fudan University. For the construction of FaDu tumor-bearing models, each male BALB/c nude mouse was received subcutaneous injection of approximately 1×106 FaDu

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cells in 100 µL of PBS in the right buttock. Tumor volume was approximate by the following equation: Tumor volume=0.5 × (tumor length) × (tumor width)2.

2.2. Preparation of nanogels. The biodegradable PSBMA nanogels were synthesized by reflux precipitation copolymerization of SBMA and BAC at different mass ratio in 40 mL of acetonitrile (AN) using 4 mg of AIBN as the initiator (Table 1). Briefly, the mixture was dispersed under ultrasound for 5 minutes, stirred at 200 rpm in nitrogen atmosphere, and incubated at 100 oC for 1 hour. After polymerization, the resulting nanogels were spun down at 5000 × g for 15 min and washed three times with deionized water, resuspended in deionized water, and stored at 4 oC for further use. The non-biodegradable nanogels (nPSBMA nanogels) were prepared using the same method as biodegradable PSBMA nanogels except that BAC was replaced with 60 mg of MBA and mixed with 140 mg of SBMA to acquire nanogels with similar hydrodynamic size.

Table 1. The controllable recipes and colloidal properties of PSBMA nanogels Samples

SBMA

BAC

AIBN

AN

Dh

PDI

ZP

(mg)

(mg)

(mg)

(mL)

(nm)

PSBMA-10

180

20

4

40

290

0.128

-4.0 ± 0.4

PSBMA-20

160

40

4

40

252

0.031

-7.9 ± 0.8

PSBMA-30

140

60

4

40

236

0.018

-10.2 ± 1.0

PSBMA-40

120

80

4

40

231

0.002

-13.5 ± 1.3

(mV)

Dh: Hydrodynamic diameters of nanogels measured in phosphate buffer of pH 7.4 at 37 °C (1 mg/mL). PDI: Polydispersity index of the particle size. ZP: Zeta potential of nanogels in phosphate buffer of pH 7.4 at 37 °C (1 mg/mL). The PEGylated biodegradable nanogels (POEGMA nanogels) were obtained through an aqueous precipitation polymerization process as we previously reported.28 7



2.3. Colloid stability and biodegradable property of nanogels. To test the colloid stability of nanogels, PSBMA nanogels were mildly mixed with 10 % fetal bovine serum in phosphate buffered saline (PBS). The stability of nanogels was detected by monitoring the hydrodynamic size by dynamic scattering light (DLS) after different periods of incubation. The degradation behavior of PSBMA nanogels was observed by tracking the change of turbidity in the presence of GSH with different concentrations and compared with the nanogels without GSH. The turbidity of nanogels was decreased with the degradation of cleavable disulfide linkage which could be monitored by DLS. The degraded polymer molecular weight was further analyzed using a gel permeation chromatography (GPC) system (HP Agilent series 1100).28

2.4. In vitro drug loading and release study. DOX, as the model antitumor drug was encapsulated into the biodegradable nanogels to study the drug release behaviors. For DOX loading in nanogels, 3 mg of DOX was dissolved in 3 mL of deionized water, and the solution pH was adjusted to 8.0 with 0.1 M NaOH. Afterward, 10 mg of nanogels was added in the DOX solution, and stirred 24 h at room temperature. The nanogels were then spun down at 5000 × g for 20 min, and washed two times using deionized water to remove the unloaded drug. The supernatant was collected and determined the DOX concentration by UV-visible spectrometry (Perkin-Elmer, Lambda750, USA) at the excitation wavelength of 488 nm. The DOX loading content (LC) and drug encapsulation efficiency (EE) were calculated according to the following formulations: LC(%) = (DOX loaded in nanogels) / (total nanogels weight); EE(%)=(DOX loaded in nanogels)/(DOX input). To obtain the DOX release behaviors, 2 mL of DOX-loaded nanogels containing 0.2 mg of DOX was sealed in a 14000 Da dialysis bag and incubated in 100 mL of phosphate buffer saline (pH7.4) containing different concentrations of GSH (0 and 10 mM) at 37 ◦C under oscillation. At different time points, 2 mL of release solution was removed and replaced with 8


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an equal volume of fresh solution. The total amount of DOX released from nanogels was obtained through UV−visible spectrometry. The cumulative released drug was calculated as percentage of the total drug loaded in nanogels and plotted with time.

2.5. Cytocompatibility and cytotoxity assay of nanogels. The cytocompatibility of PSBMA nanogels was evaluated by MTT assay. Briefly, the human hypopharyngeal carcinoma cell lines (FaDu) were seeded on 96-well plates (1×104 cells/well) and incubated in MEM supplemented with 10% FBS for 24 h. Afterward, cells were treated with blank PSBMA or n-PSBMA nanogels at the concentration range of 0 - 200 µg/mL for 24 h. Then, 20 µL of MTT solution (5 mg/mL) was added and incubated for 4 h. After that, the supernatant was removed and 150 µL of dimethylsulfoxide was added into each well. The plate was mildly oscillated and detected by an automated microplate spectrophotometer (EpocH2, BioTek Instruments, USA) at 490 nm. The cytotoxicity of DOX-loaded PSBMA nanogels (PSBMA-DOX) and DOX-loaded nPSBMA nanogels (n-PSBMA-DOX) against FaDu cells were also evaluated by MTT assay.

2.6. Cellular uptake of nanogels. The cellular uptake of nanogels was investigated on FaDu cells. Approximately 1×106 cells per dish were seeded in confocal dishes (Corning, New York, USA) and incubated in cell culture medium for 24 h. Afterward, cells were incubated with free DOX, PSBMA-DOX or POEGMA-DOX (DOX-loaded POEGMA nanogels) at the DOX concentration of 5 µg/mL for 1 or 4 h. Then cells were rinsed, stained with 4',6-diamidino-2-phenylindole (DAPI), and observed with a Nikon laser confocal microscope (C2plus, Nikon, Japan). The cellular uptake of PSBMA-DOX was investigated quantitatively by flow cytometry. Briefly, FaDu cells were seeded on 6-well plates at a density of 1×105 cells per well and put into incubator for 24 h. Then, cells were incubated with PSBMA-DOX at the DOX 9



concentration of 5 µg/mL for 1 or 4 h. After that, cells were washed three times with PBS, harvested by digestion with trypsin, spun down at 800 × g for 5 min, resuspended in PBS, and then subjected to flow cytometry (Beckman Coulter, Gallios, USA).

2.7. In vivo near-infrared fluorescence (NIRF) imaging of nanogels. To evaluate tumor delivery of nanogels by in vivo NIRF imaging, nanogels were firstly labeled with a NIRF dye, Cyanine7.5. Briefly, 25 mg of PSBMA or POEGMA nanogels were stirred with 0.2 mg of TCEP for 12 h to generate free thiol by partial reduction of disulfide bonds in nanogels. The thiol-modified nanogels were stirred with 5 mg of Sulfo-Cyanine7.5 maleimide for 24 h under nitrogen atmosphere to fabricate Cyanine7.5-labeled nanogels (PSBMA-Cy7.5 or POEGMACy7.5) via thiol-maleimide reaction.28 For in vivo NIRF imaging of nanogels, FaDu tumor-bearing mice were intravenously injected with 100 µL of free Cy7.5, PSBMA-Cy7.5 or POEGMA-Cy7.5 at a Cy7.5 dose of 20 µg and subjected to in vivo fluorescence imaging with an optical small animal imaging system (Bruker MI, In Vivo Xtreme, USA) at different time points after injection. NIFR images were acquired using a single-filter set with excitation of 790 nm and emission of 830 nm.

2.8. In vivo biodistribution of nanogels. Biodistribution of nanogels was investigated in FaDu tumor-bearing nude mice to evaluate tumor delivery of PSBMA nanogels. When the tumor volume reached ~200 mm3, mouse models were randomly divided into two groups and intravenously injected with 100 µL of PSBMA-Cy7.5 and POEGMA-Cy7.5 at a dose of 1 mg/mL, respectively. At 24 or 48 h post injection, three mice from each group were sacrificed, perfused with saline. Major visceral organs including hearts, livers, spleens, lungs, kidneys, and tumors were collected, weighed, homogenized in 2 mL of PBS, and then centrifuged at 1200 rpm for 10 min. The nanogels concentration in the supernatant of each sample was

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measured using a fluorescence lifetime spectrometer. The distribution of nanogels in organs was displayed as the percentage of injected dose per gram of tissue (% ID/g).

2.9. In vivo pharmacokinetics of nanogels. Ten male BALB/c mice (aged 5-6 weeks) were randomly divided into two groups and administrated with PSBMA-Cy7.5 and POEGMA-Cy7.5 (1 mg/mL, 100 µL) via tail vein injection, respectively. 50 µL of orbital venous blood from each mouse was collected at different time points post injection (0.5, 1, 2, 4, 8, 12, 24, 48 h). Five days later, the mice in each group received a second injection of Cy7.5-labeld nanogels at the same dose as the first injection. Blood collection was performed as described above. All the blood samples were dissolved in 2450 µL of PBS solution containing 3 IU of heparin sodium, and centrifuged at 1200 × g for 10 min. The nanogel concentration in the supernatant of each sample was measured using a fluorescence lifetime spectrometer (QM-40, Photo Technology International, USA) and plotted with time. The pharmacokinetics parameters were calculated by fitting the pharmacokinetics curve to a twocompartment model.

2.10. Immunogenic response of nanogels. Immunogenic response of nanogels was investigated by measuring the immunoglobulin M (IgM) and immunoglobulin G (IgG) levels in blood from the mice injected with nanogels. In brief, male BALB/c mice weighed 20 ± 4 g were randomly divided into three groups: Control, PSBMA, and POEGMA group. For the PSBMA, and POEGMA group, mice received an injection of PSBMA nanogels and POEGMA nanogels at the dose of 1 mg/kg, respectively. Mice in the Control group received an injection of an equal volume of saline. Five days after injection, 1 mL of blood was collected from each mouse through orbital sinus (n=5 per group) and serum was prepared for IgM measurement according to the ELISA protocol. Seven days after the first injection, the remaining mice in the three groups received the second injection of PSBMA nanogels, 11



POEGMA nanogels at the dose of 1 mg/kg, and an equal volume of saline, respectively. Five days after the second injection, 1 mL of blood was collected from each mouse for IgG measurement (n=5 per group).

2.11. In vivo antitumor efficacy. Twenty-five FaDu tumor-bearing nude mice were randomly divided into five groups (n=5). When tumor volume of mouse models reached approximately 100 mm3, mouse models received different treatments and the day when the treatments initiated was regarded as day 0. Free DOX, n-PSBMA-DOX, POEGMA-DOX, and PSBMA-DOX were injected into 4 groups of mouse models via tail vein on day 0 and day 5 at a DOX dose of 5 mg/kg body weight, respectively. NS was administrated to mice in the control group at a volume of 100 µL on day 0 and day 5. We measured the tumor size and body weight of the mice every other day. On the day 15, all the mice were euthanized, and tumors were dissected and weighed. Histological analysis was performed on tumors and major visceral organs by hematoxylin and eosin (H&E) staining. TUNEL assay were performed to detect apoptotic cells in the tumor slices.

2.12. Blood biochemistry and blood routine test. To test the nanogels in vivo biosafety, twenty-four male BALB/c mice weighed 20-22 g were randomly divided into three groups as Control, PSBMA, and POEGMA group. For PSBMA and POEGMA group, 100 µL of PSBMA and POEGMA nanogels at the dose of 1 mg/mL was administrated via tail vein, respectively. The experimental details of blood routine indicators were provided in our previous work.29

2.13. Statistical analysis. Statistical differences were analyzed by GraphPad Prism 5. Unpaired student’s t-test was used for assessment of statistically significant differences between two groups. One way ANOVA was performed for multi-group comparison. P values < 0.05 were considered significant difference and ns denoted no significant difference. Data were expressed as means ± SD. 12


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3. RESULTS AND DISCUSSION 3.1. Preparation and characterization of PSBMA nanogels In this work, biodegradable PSBMA nanogels were facilely obtained by reflux precipitation copolymerization of SBMA and BAC. Reflux precipitation polymerization is a simple onestep method to obtain monodisperse hydrophilic nanoparticles without adding any surfactants.30-32 Therefore, the obtained nanogels can be easily cleaned and safely used in biomedical fields. Generally, the monomers used in this method should be readily soluble in solvent while the as-prepared polymers should become insoluble in solvent and assemble into nanoscaled particles with the molecular weight increasing, and finally form stable nanogels after crosslinked by BAC. To optimize the particle size of the nanogels, the ratio of SBMA and BAC was carefully adjusted (Table 1). It was shown that all the nanogels except PSBMA10 presented narrow size distributions with PDI

Biodegradable Zwitterionic Nanogels with Long Circulation for

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