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Design and fabrication of multifunctional sericin nanoparticles for tumor targeting and pH responsive subcellular delivery of cancer chemotherapy drugs Lei Huang, Kaixiong Tao, Jia Liu, Chao Qi, Luming Xu, Panpan Chang, Jinbo Gao, Xiaoming Shuai, Guobin Wang, Zheng Wang, and Lin Wang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.5b11617 • Publication Date (Web): 08 Feb 2016 Downloaded from http://pubs.acs.org on February 13, 2016

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ACS Applied Materials & Interfaces Page 1 of 34

Design and fabrication of multifunctional sericin nanoparticles for tumor targeting and pH responsive subcellular delivery of cancer chemotherapy drugs #

#

Lei Huang1, , Kaixiong Tao2, , Jia Liu1, Chao Qi1, Luming Xu1, Panpan Chang3, Jinbo Gao2, *

*

Xiaoming Shuai2, Guobin Wang2, , Zheng Wang1,2, , Lin Wang1,4,

*

1

Research Center for Tissue Engineering and Regenerative Medicine, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 2

Department of Gastrointestinal Surgery, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 3

Medical Research Center, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022. 4

Department of Clinical Laboratory, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 430022.

# *

These authors contributed equally to this work. Correspondence to: Lin Wang, Phone: 86-27-85726612. E-mail: [email protected] Zheng Wang, Phone: 86-27-85726612. E-mail: [email protected]

Or to Guobin Wang, Phone: 86-27-85726612. E-mail: [email protected]

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ABSTRACT The severe cytotoxicity of cancer chemotherapy drugs limits their clinical applications. Various protein-based nanoparticles with good biocompatibility have been developed for chemotherapy drug delivery in hope of reducing drugs’ side effects. Sericin, a natural protein from silk, has no immunogenicity and possesses diverse bioactivities that have prompted sericin’s application studies. However, the potential of sericin as a multi-fucntional nanoscale vehicle for cancer therapy have not been fully explored. Here we report the successful fabrication and characterization of folate-conjuguated sericin nanoparticles with cancer-targeting capability for pH-responsive release of doxorubicin (these nanoparticles are termed “FA-SND”). DOX is covalently linked to sericin through pH-sensitive hydrazone bonds that render a pH-triggered release property. The hydrophobicity of DOX and the hydrophilicity of sericin promote the self-assembly of sericin-DOX (SND) nano-conjugates. Folate (FA) is then covalently grafted to SND nano-conjugates as a binding unit for actively targeting cancer cells that overexpress folate receptors. Our characterization study shows that FA-SND nanoparticles exhibit negative surface charges that would reduce non-specific clearance by circulation. These nanoparticles possess good cytotoxicity and hemocompatibiliy. Acidic environment (pH 5.0) triggers effective DOX release from FA-SND, 5-fold higher than does a neutral condition (pH 7.4). Further, FA-SND nanoparticles specifically target folate-receptor rich KB cells, and endocytosed into lysosomes, an acidic organelle. The acidic microenvironment of lysosomes promotes a rapid release of DOX to nuclei, producing cancer specific chemo-cytotoxicity. Thus, FA-mediated cancer targeting and lysosomal-acidity promoting DOX release, two sequentially-occurring cellular events triggered

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by the designed components of FA-SND, form the basis for FA-SND to achieve its localized and intracellular chemo-cytotoxicity. Together, this study suggests that these FA-SND nanoparticles may be a potentially effective carrier particularly useful for delivering hydrophobic chemotherapeutic agents for treating cancers with high-level expression of folate receptors.

Keywords: sericin; nanoparticles; folate; pH sensitivity; doxorubicin

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INTRODUCTION Although various therapies have been developed for cancer treatment, chemotherapy remains as the most common approach. However, chemotherapy has its limitations, including severe cytotoxicity side effects, ineffective access to cancer local, and unstable blood concentration.1 The form of nanoscale drug delivery vehicles has been proposed to overcome these limitations.2-3 Nanocarriers have high drug loading capacity and can be incorporated with hydrophilic or hydrophobic drugs. Importantly, they can be designed to possess “targeting” capability to cancer cells through active targeting via nanocarrier-conjugated ligands that specifically bind to molecules in cancer cells. With respect to materials options for nanocarriers, natural polymers have drawn attention because of their excellent biocompatibility, in vivo biodegradability, and abundant renewable source.4 Their versatile chemical structures allow them to be conveniently functionalized with tumor targeting ligands and stimuli-responsive groups towards the design of intelligent drug delivery systems.5 Such systems release cargoes to tumor sites through cleavage of chemical bonds upon the presence of expected physiological or external stimuli, such as pH,6-7 temperature, 8

or enzymatic activity 9 within tumor microenvironment. Silk sericin, a natural protein derived from silkworm cocoons, acts to glue fibroin fibers

together to form silk fibers. Sericin is a hydrophilic macromolecular protein containing abundant polar side chains made of hydroxyl, carboxyl and amino groups that provide sericin with high chemical reactivity. Sericin has no immunogenicity10-16 and possesses diverse bioactivities and excellent biocompatibility with cells and tissues.17-18 Thus, various sericin formulations for

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biomedical applications have been explored, including hydrogels,19 films,20 3D scaffolds,21 and microparticles.22 In the field of nanocarriers, two types of self-assembled sericin nanoparticles, sericin-PEG23 and sericin-poloxamer nanoparticles,24 and a type of desolvated sericin nanoparticles,25 have been reported for drug and gene delivery. However, the presence of non-biodegradable poloxamer and drug leak due to the lower micelle stability26 limit their applications in vivo. Moreover, all these reported sericin nanoparticles lack the ability of targeting tumors in a specific manner. Thus, multifunctional sericin nanoparticles with tumor specific targeting and controlled drug release might be valuable for cancer therapy. Taking advantage of high expression of folate receptors in many human tumor cells,27 we propose to fabricate sericin nanoparticles with active tumor targeting capability by conjugating folate with the nanoparticles, which would allow sericin nanoparticles to be preferentially endocytosed by tumor cells via receptor-mediated endocytosis. Since this endocytosis trafficks nanoparticles into intracellular endo-lysosomal compartments that are acidic (low pH), we design hydrozone bonds, a pH-responsive chemical bonds, to connect DOX and sericin, thus conferring a controlled drug release feature to this nanoscale delivery system. This covalent link may also help reduce the possible drug leak. Given the hydrophilic nature of sericin and the hydrophobic nature of DOX, DOX-sericin conjugates can self-assemble forming nanoparticles. This strategy would facilitate the effective intracellular release of DOX, which might improve anti-tumor efficacy of this sericin nanoparticle system. Using this design, we have successfully fabricated folate-sericin-DOX (FA-SND) based nanoparticles and characterized these FA-SND nanoparticles, including size, zeta potential,

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pH-triggered drug release and hemocompatibility. Their subcellular localization, intracellular drug release, and cell targeting specificity are evaluated. Our study demonstrates the potentiality of FA-SND nanoparticles as a drug delivery vehicle for cancer therapy.

EXPERIMENTAL SECTION Materials: Silkworm cocoons (Bombyx mori, baiyu) were kindly given by the Sericultural Research Institute, China Academy of Agricultural Sciences (Zhenjiang, Jiangsu, China). 4-morpholineethanesulfonic

acid

(MES),

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide

hydrochloride (EDC·HCl), Bafilomycin A1 and N-hydroxysulfosucnimide sodium salt (sulfo-NHS) were purchased

from

Sigma

Chemical

(St,

Louis,

MO).

Doxorubicin

hydrochloride (DOX·HCl) was obtained from Meilun Biology Technology Co. Ltd (Dalian, China). Folate (FA), dimethyl sulfoxide (DMSO), and hydrazine hydrate (N2H4·H2O, 85%) were purchased from Sinopharm Chemical Reagent Co. Ltd (Shanghai, China). The human cervical cancer cell line (HeLa), the mouse myoblast cell line (C2C12) and the human oral epithelium carcinoma cell line (KB) were obtained from the China Center for Type Culture Collection at Wuhan University (Wuhan, China). The cells lines were cultured in folate (FA) free RPMI 1640 or high glucose DMEM medium supplemented with 10% (v/v) fetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin at 37°C in 5% CO2 incubators. Isolation of silk sericin: Sericin protein of silkworm cocoons (Bombyx mori, baiyu) was extracted according to the methods used in alkaline degumming process with modifications.21 Briefly, cocoons (1.0 g) were cut into pieces and boiled in 0.02 M solution of sodium carbonate

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(100 mL) at 100 ºC for 60 minutes. The degumming solution (supernatant solution) was obtained by centrifugation and filtration to remove the impurities, then dialyzed (cellulose dialysis membranes, MWCO 8-14 kDa, Spectrum Laboratory, Inc., USA) against deionized water for 2 days at room temperature. Subsequently, the sericin powder was obtained by lyophilization and kept in refrigerator before use. Synthesis of sericin-hydrazine conjugates (SNH): Hydrazine functionalized sericin (SNH) was synthesized through the condensation reaction between the carboxyl group of sericin and hydrazine hydrate. Sericin powder (600 mg) was completely dissolved in MES solution (pH 5.0, 60 mL). EDC·HCl (3.83 g, 20 mmol) and sulfo-NHS (1.09 g, 5 mmol) were added to the sericin solution with stirring. Thereafter, 1.0 mL hydrazine hydrate (85%, v/v) was added dropwise to the mixture. This reaction was carried out at room temperature for 24 h in darkness. The mixture was dialyzed (cellulose dialysis membranes, MWCO 3.5 kDa, Spectrum Laboratory, Inc., USA) against deionized water for 2 days to remove unreacted reagents or impurities, and followed by lyophilization to obtain SNH conjugate powder. The yield of SNH was 80%. Synthesis of sericin-DOX conjugates (SND): DOX was grafted onto SNH conjugates via the hydrazone bond according to the methods previously reported.28-29 Briefly, SNH (100 mg) was dissolved in 10 mL anhydrous dimethyl sulfoxide (DMSO), and followed by the addition of triethylamine (TEA, 20 µL) and DOX·HCl (10 mg). The mixture was stirred at room temperature for 24 h in darkness. Subsequently, the reaction solution was dialyzed (MWCO 3.5 kDa) against deionized water replaced every 12 h for 3 days to remove the excess drug and DMSO in the dark. The thin layer chromatography (TLC) showed that DOX spot had a significantly higher

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retardation factor (Rf) value than did SND (Figure S2B) when visualized by both bright field and 254-nm light, suggesting the free DOX that was not associated with the SND could be readily removed by dialysis. Finally, the sericin-DOX conjugate was obtained by lyophilization. The yield of SND was 90%. Preparation of folate-sericin-DOX conjugates (FA-SND): The conjugation of folate to sericin-DOX conjugates was performed according to the method previously reported.6 Briefly, folate (5 mg) was dissolved in 1 mL anhydrous DMSO, and followed by the addition of EDC·HCl (3 mg) and sulfo-NHS (2 mg) under stirring at room temperature in darkness. After stirring for 3 h to completely activate the carboxyl groups of folate, the DMSO mixture was added dropwise to the sericin-DOX solution (3 mL, 20 mg/mL) and stirred for 10 h at room temperature in darkness. The mixed solution was dialyzed (MWCO 3.5 kDa) against deionized water for 3 days to remove the free folate and DMSO in darkness. Finally, the solution was lyophilized to obtain FA-SND that was then kept in refrigerator before use. The yield of FA-SND was 81%. The folate content in FA-SND was 0.156 mmol/g determined by UV-vis absorbance spectra at 350 nm. Characterization of copolymers: The ultraviolet-visible absorbance spectra of sericin, folate, DOX·HCl, SND, and FA-SND were recorded using a NanoDrop-photometer (Thermoscientific, USA). The amount of conjugated DOX on FA-SND was within the range of 3.0 to 5.0 wt%, determined by measuring the absorbance peak at 490 nm (subtracting the absorbance of sericin at that wavelength) based on a standard curve of DOX. Fourier transform infrared (FTIR) spectra analysis was carried out using an FTIR spectroscopy (Nexus, Thermal Nicolet, USA). The

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ninhydrin (NHN) colorimetric assay was performed to determine the content of the hydrazide groups attached to SNH by the previously established method.19 Briefly, the ninhydrin solutions were prepared as previously described 14. SNH solution (1 mL, 10 mg/mL) was mixed with 0.5 mL solution A (20 mL of 0.067 mol/L Na2HPO4 mixed with 5 mL of 0.067 mol/L KH2PO4) and 0.5 mL solution B (2 g ninhydrin and 80 mg SnCl2 were added into 5 ml H2O, stirred for 30 min, then stored in a dark bottle for 24 h.), heated to 100 ºC in water bath for 15 min. The absorbance of the solution at 570 nm was employed to assess the content of the hydrazide groups in SNH, which was measured with a spectrophotometer (Infinite F50, Tecan, Switzerland) after the solution cooled down to room temperature. The sericin solutions were used as the controls. The molecular weight profiles of sericin and SNH were examined by the polyacrylamide gel electrophoresis (SDS-PAGE) as previously described.30 Consistent with previous studies,17, 22 the sericin obtained from this method was degraded containing the large amount of polypeptides with low molecular weight ranging from 25 to over 170 KDa (Figure S1). Size, morphology, and zeta potential: The sericin, SNH, SND and FA-SND were diluted in pure water to a final polymer concentration of 100 µg/mL. Hydrodynamic diameter and zeta potential were determined by a Zetasizer Nano-ZS 3600 (Malvern Instruments, UK). Each sample was measured in three experimental repeats to obtain an average value. The morphology of sericin, SNH, SND and FA-SND was imaged using an atomic force microscopy (AFM, Nanoscope Ⅲa, Veeco, USA). The samples were diluted in ultrapure warter (100 µg/mL), dropped onto mica surface and dried in air at room temperature overnight before measurement. Sizes of SND and FA-SND were quantitatively measured by ImageJ software (version 1.48v,

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NIH). In order to determine the stability of FA-SND in aqueous solution, hydrodynamic diameter distribution analysis was performed in pure water, in phosphate buffered saline (PBS), and in PBS supplemented with 10% FBS at 37ºC using a Zetasizer Nano-ZS 3600 (Malvern Instruments, UK). Hemolysis assay:

Red

blood

cells

(RBCs)

were

collected

from

5 mL of human blood sample anticoagulated with EDTAK2 by centrifuging at 1000 rpm for 5 min, followed by washing with PBS. RBCs were diluted in PBS solution (2%, v/v), and 1 mL of the suspension was added to 1 mL of FA-SND nanoparticles suspended in PBS at the final concentrations of 200, 500 and 1000 µg/mL. Meanwhile, PBS and 1% (v/v) TritonX-100 were used as the negative and positive controls, respectively. The mixtures were incubated at 37 °C for 4 h, and then centrifuged at 12,000 rpm for 5 min. Subsequently, the absorbance of the supernatant

was

measured

at

545 nm

using

a

Thermoscientific

NanoDrop

2000

Spectrophotometer. The percentage of hemolysis was calculated by dividing the sample absorbance

by

the

absorbance

of

1%

Triton

X-100.31

All of the

hemolysis experiments were carried out in triplicates. In vitro drug release: The pH-responsive drug release from the FA-SND in vitro was evaluated using the dialysis method. Briefly, FA-SND (50 mg) nanoparticles were suspended in 2 mL phosphate buffer (pH 7.4) or acetate buffer (pH 5.0), and transferred into a dialysis bag (MWCO 3.5 kDa). Then, the dialysis tubes were immersed into 25 mL of PBS or acetate buffer, respectively, and stirred at 200 rpm at 37°C. At the predetermined time points, 20 µL of external media was taken and analyzed by a Thermoscientific NanoDrop 2000 Spectrophotometer at 490

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nm. The release of free DOX as a control from the dialysis bag was analyzed under the same conditions. Western blot analysis. All cell lysates were prepared and quantified using a BCA protein assay kit (Bio-Rad, USA). Protein samples (80 µg) were separated in 12% acrylamide gel and electrophoretically transferred to a PVDF membrane. Membranes were blocked with 5% skim (non-fat) milk and then probed with folate receptor 1 polyclonal antibody (1:1000, Proteintech, China), followed by the incubation with a horseradish peroxidase (HRP) conjugated secondary antibody. The protein bands were visualized using chemiluminescent HRP substrate (Millipore, Billerica, MA) and observed in a chemiluminescence system. Cytotoxicity assay: The cytotoxicity of sericin protein polymers against the HeLa cells was assessed using a Cell Counting Kit-8 kit (CCK-8 kit, Dojindo laboratories, Kumamoto, Japan) according to the manufacturer’s instructions. HeLa cells were seeded at the density of 5 × 103 cells/well in 96-well plates. After 24 h, the cells were treated with sericin or SNH at different concentrations. After incubation for another 48 h, the viability of cells was measured via CCK-8 assay. Cell viability within each group was indicated as a percentage of the viability of non-treated cells. To determine how much FA-targeting and pH-sensitive drug release contributed to decreased cell viability, the cytotoxicity of FA-SND nanoparticles to KB cells in the presence of FA (1 mM) or NH4Cl (20 mM) for 48 h was evaluated using CCK-8 assay. KB cells were seeded to 96-well plates at the density of 104 cells per well. After 24 h, cells were incubated with FA-SND at final doxorubicin concentrations of 0.5, 1.0, and 2.0 µg/mL with or without the addition of NH4Cl (20

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mM) and folate (1 mM), respectively. After 48 h, the CCK-8 assay was measured as described above. The cell viability of the FA-SND treatment with or without folate or NH4Cl was determined as a percentage of the viability of non-treated cells (control). Intracellular uptake study: The cellular uptake behavior of FA-SND was analyzed via fluorescence microscopy and flow cytometry. For fluorescence microscopy imaging, KB cells and C2C12 cells were seeded to 6-well plates at the density of 105 cells/ well. After incubation for 24 h, the medium was replaced and the cells were treated with FA-SND where DOX concentration was 10 µg/mL. For folate blocking experiments, the KB cells were treated with FA-SND in the medium containing 1 mM free folate. After incubation for 3h, the cells were rinsed three times with PBS and fixed by 4% paraformaldehyde for 15 min. Then, the cell nuclei were stained with 4’, 6-diamidino-2-phenylindole (DAPI). The samples were imaged using a fluorescence microscope (Olympus, Japan). For the flow cytometry analysis, KB cells were seeded in 6-well plates at the density of 2×105 cells per well for 24 h. The cells were treated with FA-SND and FA-SND in the presence of 1 mM free folate (each treatment with the same concentration of DOX (20 µg/mL)). Non-treated cells were used as a control. After 30 min or 3 h, the cells were washed three times with PBS, harvested using a trypsin-EDTA solution, and suspended in PBS containing 1.0% glutaraldehyde. The FACS analysis was performed to analyze the DOX fluorescence intensity using Calibur (Becton Dickinson, USA). Analysis of intracellular distribution of FA-SND nanoparticles by confocal laser-scanning microscopy: KB cells were seeded onto glass coverslips placed in 6-well plates at the density of 105 cells per well overnight. The cells were treated with FA-SND where DOX concentration was

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10 µg/mL for 4 h or 16 h, respectively, or with free DOX (10 µg/mL) for 4 h as a control. The cells were then washed with PBS, stained with 250 nM of Lysotracker Green DND-26 (Molecular Probes, USA) for 30 min at 37 ºC, followed by rinse three times with PBS. Subsequently, the cells were fixed by incubation with 4% paraformaldehyde for 15 min and stained with DAPI. Confocal laser scanning microscopy (Nikon A1Si, Japan) was performed to image the cells. Analysis of pH-sensitive drug intracellular release from FA-SND nanoparticles by confocal laser-scanning microscopy: KB cells were seeded onto glass coverslips at the density of 105 cells per well overnight. The cells were incubated with FA-SND where DOX concentration was 10 µg/mL for 24 h. NH4Cl (20 mM) and Bafilomycin A1 (1 µM) were used to neutralize the acidity of lysosomes. The cells were then washed with PBS and stained with DAPI and observed by the confocal laser-scanning microscope (Nikon A1Si, Japan). To quantitatively measure DOX release to nuclei, ImageJ software (version 1.48v NIH) was used to analyze nuclear DOX fluorescence intensity. Statistical analysis: Each experiment was performed at least three times. Experiments of the CCK-8 assay were run in 5 replicates per test sample. Comparisons between groups were analyzed using Student’s t-tests. All data were presented as mean ± SD with p < 0.05 indicating statistical significance.

RESULTS AND DISCUSSION Characterizations of folate-sericin-DOX (FA-SND) nanoparticles. The FA-SND conjugate

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was designed and synthesized as shown in Figure 1A. Due to the abundant glutamic acid and aspartic acid of sericin (Glu and Asp, 24%),22,

30

the primary amino content of sericin was

increased by approximately 3 folds (Figure S2A) after carboxyl groups were functionalized with hydrazine groups. This sericin derivative was termed SNH. The ultraviolet-visible (UV-vis) absorption spectrum analysis (Figure 2A) suggests the successful synthesis of the FA-SND conjugate as the characteristic absorption peaks of DOX, FA and sericin at 490 nm, 350 nm, and 280 nm, respectively, were detected.6

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Figure 1. Schematics showing the fabrication of FA-SND nanoparticles and the mechanisms of their cancer targeting and pH-responsive intracellular drug release. (A) Schematics for synthesis and self-assembly of FA-SND (folate-conjugated sericin covalently linked with DOX, FA-SND) based nanoparticles. Sericin was functionalized with hydrazine groups via carbodiimide reactions. DOX (red) was then grafted to hydrazine-functionalized sericin (SNH) through acid-sensitive hydrazone bonds, thus forming DOX-conjugated sericin (SND). The attachment of hydrophobic DOX onto hydrophilic sericin induces amphiphilic conjugation resulting in the formation of nanoaggregates in aqueous media. Finally, FA-SND conjugates were synthesized via a reaction between sericin’s primary amines and the N-hydroxysuccinimide ester of folate. The hydrophobic folate grafting to the hydrophilic amines of the surface of SND induces the formation of FA-SND nanoaggregates. (B) Schematics showing that FA-SND nanoparticles were endocytosed by cells through folate-receptors mediated endocytosis. Endocytosed nanoparticles were delivered into endosomes and lysosomes where low pH triggered intracellular DOX release from FA-SND nanoparticles.

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The sericin derivatives and FA-SND conjugates were further characterized by FTIR spectra (Figure 2B). Sericin had its characteristic amide peaks at 1655 cm-1 (amide I; due to C=O stretching), 1540 cm-1 (amide II; deriving from N-H in-plane bend), and 1243 cm-1 (amide III; deriving from C-N stretch) (Figure 2B), consistent with previous reports.19, 21 In addition to these sericin’s specific peaks, SNH displayed two extra new absorption bands at 1043 cm-1 (vs(C-N)), and 953 cm-1 (δ(N-N)) (Figure 2B), which were likely attributed to the existence of hydrazide bonds (-CONHNH2),29 suggesting the successful grafting of the hydrazine linkers to the sericin backbone. The FTIR spectrum of sericin-DOX (SND) conjugates had small shoulders at 1410, 1122, 1020, and 988 cm-1 (Figure 2B), possibly due to the different quinone and ketone carbonyls of DOX. 32 After folate was grafted to SND, the new peaks at 1610 cm-1 (C=O stretching) and 1566 cm-1 (N-H in-plane bend) appeared in the spectrum of FA-SND. These new peaks were likely caused by the amino bonds formed between the carboxyl groups of FA and the primary amines of SND.33

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Figure 2. Characterizations of FA-SND nanoparticles. (A) Ultraviolet-visible absorbance spectra of FA-SND, DOX, sericin, SND, and FA in pure water. (B) The FTIR spectra of sericin, SNH, SND, and FA-SND. (C) The atomic force microscopy (AFM) images of SND nanoparticles (upper panel), and FA-SND nanoparticles (lower panel). (D) Size distribution of FA-SND, SND, SNH, and sericin particles was analyzed by dynamic light scattering (DLS) (n=3 experimental repeats). (E) Zeta potential of SND and FA-SND nanoparticles in pure water at 37 ºC measured by DLS.

Attachment of hydrophobic moieties to hydrophilic polymers has long been proposed to construct nano-sized supramolecular self-assemblies.5,

34

SND were self-assembled into

nanoparticles, which was confirmed by atomic force microscopy (AFM). The SND nanoparticles were spherical with the average size of 35.8 ± 7.8 nm (n=100) (Figure 2C). Compared to sericin (120 nm) and SNH nanoparticles (10 nm) measured by dynamic light scattering (DLS) (Figure 2D and S3), SND nanoparticles were medium in size, 42.5 ± 4.1 nm (Figure 2D), which was

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slightly larger than that measured by AFM, likely because the SND nanoparticles were hydrated for DLS analysis while dried during the sample preparation for AFM analysis. The FA-SND nanoparticles retained the spherical shape (Figure 2C) with the average size of 53.8 ± 17.6 nm (n=80), which was larger than that of SND. The zeta potential of SND and FA-SND nanoparticles were 5.21 ± 0.36 mV and -15.15 ± 1.51 mV, respectively, indicating that folate conjugation decreases surface charges (Figure 2D), likely because the consumption of the amino groups resulted in a higher ratio of carboxyl groups to amino groups on the surface of FA-SND. Given the slightly negative surface charges of FA-SND, we further assessed the stability of FA-SND in PBS only and in PBS supplemented with 10% FBS mimicking in vivo conditions at 37 °C by measuring particle sizes using DLS. The size distribution of FA-SND nanoparticles in PBS was nearly identical to that in water (Figure S4A) with no obvious size change observed during a 24-hour incubation in PBS (Figure S4B). Although the mean hydrodynamic size increased from 63.4 ± 5.2 nm to 196.3 ± 5.0 nm in PBS with 10% FBS (Figure S4A), it was still within a nanoscale range, revealing a relatively good stability of FA-SND nanoparticles in the presence of FBS. The observed size increase might be caused by FA-SND’s slight aggregation possibly due to FA-SND’s negative surface charges and hydrophobic molecule conjugation. Particle size and surface zeta potential are two key factors affecting circulation time, biodistribution, and therapeutic efficacy of polymeric nanoparticles.35-36 The sub-100nm range is known to favor particle accumulation in solid tumors through an enhanced permeability and retention (EPR) effect.2 As near-neutral surface potential (-15 to 10 mV) helps nanoparticles avoid clearance and promotes local accumulation in target tissue in vivo,37-38 slightly negative

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surface charges of FA-SND nanoparticles may facilitate their retention in local, which might improve their antitumor efficacy.

Cytotoxicity and Hemolysis of FA-SND nanoparticles. Cytotoxicity of nanomaterials is critical for their biomedical applications.39 After sericin was functionalized with hydrazine groups (SNH), no significant cytotoxicity was observed even when its concentration reached 1 mg/mL (Figure 3A). Consistent with SNH’s low cytotoxicity, FA-SND nanoparticles did not induce hemolysis at all the concentrations tested (0.2-1 mg/mL) (Figure 3B). These results suggest that sericin derivatives and FA-SND nanoparticles have low cytotoxicity and might not cause hemolysis in vivo.

Figure 3. Cytotoxicity and hemolysis of sericin and its derivatives. (A) The cytotoxicity of sericin and drug-free SNH after being incubated with HeLa cells at different concentrations for 48 h was evaluated using CCK-8 assay. n = 5 experimental repeats per group. (B) Hemolysis assay of FA-SND nanoparticles at different concentrations (negative control, phosphate buffer solution (PBS); positive control, 1% (v/v) Triton X-100).

pH-responsive DOX release from FA-SND nanoparticles. To examine the release efficiency of DOX from FA-SND nanoparticles, FA-SND nanoparticles were incubated in phosphate buffer

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solution (PBS, pH 7.4) and acetate buffer solution (pH 5.0) in a dialysis bag at 37°C, respectively. The release of free DOX as a control from the dialysis bag rapidly reached the same plateau (80%) at pH 5.0 or 7.4 after a short 4-hour incubation (Figure 4A). In contrast, FA-SND nanoparticles were stable at pH 7.4 (PBS) with only 13.2% DOX released after a 52-hour incubation (Figure 4B), whereas approximately 60% DOX was released from FA-SND nanoparticles after 30 hours at pH 5.0 (acetate buffer solution), a nearly 5-fold increase (Figure 4B). These results are consistent with our design where DOX release was dependent on the cleavage of the hydrazone bonds, a type of pH-sensitive bonds,7,

40

between DOX and sericin. Therefore, FA-SND

nanoparticles would be stable in physiological conditions during circulation and release DOX after being internalized into acidic cellular organelles (pH 4.0-6.0), such as endosomes and lysosomes. Previous studies report some polymer micelle carriers for DOX delivery, such as DOX-conjugated PEG-b-poly(aspartic acid).41 In that carrier, an amido bond, a non tumor-microenvironment-responsive linkage, was employed to conjugate DOX. This conjugated DOX played a “structural” role endowing the system with amphiphilicity, but was not able to be released as a cargo drug. Free DOX was thus entrapped into these micelles as a cargo drug for delivery. However, the diffusion-dependent release of the entrapped-DOX would inevitably cause off-target toxicity to normal tissues or organs. In contrast, our system with the design of pH sensitive linkage connecting DOX and sericin could achieve their design goals while avoid their system limitations. This was because the pH sensitive linkage not only renders DOX a dual role in providing amphiphilicity to the system and acting as a cargo drug, but also offers a solution

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minimizing the non-specific DOX release.

Figure 4. The pH-responsive drug release from FA-SND nanoparticles in vitro. The release of free DOX (A) and DOX from FA-SND nanoparticles (B) at pH 5.0 (acetate buffer) and at pH 7.4 (PBS). n = 3 per group per time point.

Folate(FA)-receptor-mediated cellular uptake of FA-SND nanoparticles. Delivering chemotherapeutic agents specifically to cancer cells using cancer-targeting nanocarriers would help reduce chemotherapeutic side effects to normal cells. The efficacy of folate-directed cancer targeting of FA-SND nanoparticles was examined in KB cells, a human oral epithelium carcinoma cell line with rich expression of folate receptors. By detecting DOX fluorescence, the significant cellular uptake of FA-SND was observed in KB cells after a 3-hour incubation (Figure 5A). To test whether this uptake was dependent on FA receptors, we added free FA that would presumably compete with FA-SND to bind to FA receptors. Indeed, the addition of free FA drastically inhibited the cellular uptake of FA-SND (Figure 5A). Similarly, the flow cytometry analysis also demonstrated that free FA suppressed the cellular uptake of FA-SND by approximately 50% (Figure S5 and 5B). In contrast, FA-SND was scarcely uptaken by C2C12

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cells (Figure 5A), a mouse fibroblast cell line expressing a low level of folate receptors (Figure S6). Together, these observations indicate that FA receptors mediate the entry/uptake of FA-SND nanoparticles into KB cells.

Figure 5. Specific uptake of FA-SND nanoparticles by cancer cells expressing folate receptors. (A) The phase contrast (left column), DOX fluorescence (middle column), and their corresponding merged images (right column) of KB cells (upper two panels) treated with FA-SND in the absence or presence of 1 mM free folate (FA) for 3 h, and C2C12 (low panel) incubated with FA-SND for 3 h. DOX fluorescence indicates the existence of FA-SND. Scale bars, 50 µm. (B) Quantification of DOX mean fluorescence intensity of the KB cells treated as described in A using flow cytometry (see the corresponding flow cytometry images in Figure S5). *p < 0.01; Student’s t-tests; n = 3 experimental repeats per treatment. Intracellular distribution of FA-SND and the intracellular release of DOX from FA-SND. Efficient delivery of chemotherapy drugs to specific intracellular organelles could not only significantly potentiate their therapeutic effects but also reduce their non-specific toxicity.42 We thus examined the subcellular localization of FA-SND nanoparticles. In contrast to free DOX that is known to primarily accumulate in cell nuclei (Figure 6A), a 4-hour incubation of FA-SND with the cells led to the accumulation of DOX fluorescence in the acidic organelles-lysosomes that

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were positively stained with lysotracker (Figure 6B), which was consistent with previous studies showing that FA-conjugated nanoparticles could enter acidic endosomes or lysosomes via FA-receptor-mediated endocytosis pathways.43-44

Figure 6. Intracellular drug release from FA-SND nanoparticles examined by confocal laser scanning microscopy. (A) The confocal images showing DOX (red, 10 µg/mL) were accumulated in nuclei (blue, stained by DAPI) but not in lysosomes (green, stained by lysotracker) of the KB cells that were incubated with free DOX for 4 h. (B) DOX was mainly accumulated in the lysosomes (white arrows) of the KB cells that were incubated with FA-SND nanoparticles (10 µg/mL DOX) for 4 h. (C) DOX was accumulated in the nuclei (white arrows) 16 h after KB cells were incubated with FA-SND nanoparticles. Scale bars, 10 µm.

The confocal examination of the intracellular release of DOX showed that after a 16-hour incubation the accumulation of DOX was shifted from lysosomes to the nuclei (Figure 6C), suggesting the hydrolysis of hydrazone bonds linking DOX with FA-SND nanoparticles effectively takes place in acidic lysosomal organelles. In support of this notion, when the cells were treated with NH4Cl that neutralizes acidic microenvironment 45-46 or Bafilomycin A1, a

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specific inhibitor of the vacuolar type H+-ATPase,47 the nuclear accumulation of DOX was significantly reduced (Figure 7 and S7), indicating that the acidic condition is required for DOX release from FA-SND. Given FA-SND’s specific localization to endo-lysosomes and its pH-triggered release behavior, these nanoparticles might be able to serve as a lysosomotropic drug delivery platform.

Figure 7. pH-responsive drug release behavior of FA-SND nanoparticles at the cellular level. (A) The confocal images showing that the majority of DOX (red) was accumulated in nuclei (blue, stained by DAPI) of the KB cells that were incubated with FA-SND nanoparticles (10 µg/mL DOX) in the absence or presence of NH4Cl (20 mM) for 24 h. Scale bars, 50 µm. (B) Quantification of the nuclear DOX fluorescence intensity in the KB cells with the treatments described in A using ImageJ software. *p < 0.01; Student’s t-tests; n = 100 cells per treatment. Cancer-targeting cytotoxicity of FA-SND nanoparticles relies on their binding to FA receptors and the acidic microenvironment within endo-lysosomes. Next, we examined the tumor cytotoxicity of FA-SND nanoparticles. When being incubated with the increasing concentrations of FA-SND for 48 hours, KB cell viability was decreased in a dose dependent manner (Figure 8). When free folate was added into the media, the cytotoxicity of FA-SND was significantly reduced. Moreover, the cytotoxicity of FA-SND was also inhibited when the

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intracellular drug release was blocked by NH4Cl treatment. These results indicate that the exertion of FA-SND’s cancer cytotoxicity specifically depends on cancer cell targeting through FA-receptor-mediated endocytosis and pH-triggered intracellular drug release. These two designed features ensure targeting specificity, which would help reduce chemo-drugs’ side effects.

Figure 8. In vitro cancer-targeting cytotoxicity of FA-SND nanoparticles. The cytotoxicity of FA-SND nanoparticles to KB cells in the presence of FA (1 mM) or NH4Cl (20 mM) for 48 h was evaluated using CCK-8 assay. *p < 0.01; Student’s t-tests; n = 5 experimental repeats per treatment.

CONCLUSION We have fabricated FA-SND based nanoparticles that possess the capability of folate-directed tumor cell targeting and the property of acidic pH-responsive drug release. The FA-SND nanoparticles exhibit narrow size distribution and negative surface potential with good biocompatibility and hemocompatibility. Indeed, FA-SND nanoparticles are able to effectively target to folate-receptor positive cancer cells, be internalized into acidic organelles by FA-receptor mediated endocytosis, and release DOX within lysosomal compartments. These

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designed functions help confine the chemotherapeutic effects of FA-SND within cancer regions, thus reducing undesired chemo-drugs’ cytotoxic side effects. Given various types of abundant side chains of sericin, it is possible to further functionalize sericin nanoparticles in different ways to meet diverse therapeutic requirements, providing an adaptable, biocompatible drug delivery vehicle for personalized cancer therapy.

ASSOCIATED CONTENT Supporting Information (SI). SI section contains free amino content of sericin and SNH, and thin-layer chromatography (TLC) of SND. This materials is available free of charge via internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors Email: [email protected] Email: [email protected] Email: [email protected] Notes The authors declare no competing financial interests.

ACKNOWLEDGMENTS The work is supported by the National Natural Science Foundation of China Programs 81272559,

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81441077 and 81572866, the International Science and Technology Corporation Program of Chinese Ministry of Science and Technology S2014ZR0340, the Science and Technology Program of Chinese Ministry of Education 113044A, the Frontier Exploration Program of Huazhong University of Science and Technology 2015TS153, and the Natural Science Foundation Program of Hubei Province 2015CFA049.

REFERENCES (1) Feng, S. S.; Chien, S. Chemotherapeutic Engineering: Application and Further Development of Chemical Engineering Principles for Chemotherapy of Cancer and Other Diseases. Chem Eng Sci 2003, 58 (18), 4087-4114. (2) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nanoparticle Therapeutics: an Emerging Treatment Modality for Cancer. Nat Rev Drug Discov 2008, 7 (9), 771-782. (3) Cho, K.; Wang, X.; Nie, S.; Chen, Z. G.; Shin, D. M. Therapeutic Nanoparticles for Drug Delivery in Cancer. Clin Cancer Res 2008, 14 (5), 1310-1316. (4) Dang, J. M.; Leong, K. W. Natural Polymers for Gene Delivery and Tissue Engineering. Adv Drug Deliv Rev 2006, 58 (4), 487-499. (5) Elsabahy, M.; Wooley, K. L. Design of Polymeric Nanoparticles for Biomedical Delivery Applications. Chem Soc Rev 2012, 41 (7), 2545-2561. (6) Du, C.; Deng, D.; Shan, L.; Wan, S.; Cao, J.; Tian, J.; Achilefu, S.; Gu, Y. A pH-Sensitive Doxorubicin Prodrug Based on Folate-Conjugated BSA for Tumor-Targeted Drug Delivery. Biomaterials 2013, 34 (12), 3087-3097.

27



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(7) She, W.; Li, N.; Luo, K.; Guo, C.; Wang, G.; Geng, Y.; Gu, Z. Dendronized Heparin-Doxorubicin Conjugate Based Nanoparticle as pH-Responsive Drug Delivery System for Cancer Therapy. Biomaterials 2013, 34 (9), 2252-2264. (8) Wright, E. R.; Conticello, V. P. Self-Assembly of Block Copolymers Derived from Elastin-Mimetic Polypeptide Sequences. Adv Drug Deliv Rev 2002, 54 (8), 1057-1073. (9) Zha, Z.; Zhang, S.; Deng, Z.; Li, Y.; Li, C.; Dai, Z. Enzyme-Responsive Copper Sulphide Nanoparticles for Combined Photoacoustic Imaging, Tumor-Selective Chemotherapy and Photothermal Therapy. Chem Commun (Camb) 2013, 49 (33), 3455-3457. (10)Aramwit, P.; Kanokpanont, S.; De-Eknamkul, W.; Srichana, T. Monitoring of Inflammatory Mediators Induced by Silk Sericin. J Biosci Bioeng 2009, 107 (5), 556-561. (11)Panilaitis, B.; Altman, G. H.; Chen, J.; Jin, H. J.; Karageorgiou, V.; Kaplan, D. L. Macrophage Responses to Silk. Biomaterials 2003, 24 (18), 3079-3085. (12)Mandal, B. B.; Priya, A. S.; Kundu, S. C. Novel Silk Sericin/Gelatin 3-D Scaffolds and 2-D Films: Fabrication and Characterization for Potential Tissue Engineering Applications. Acta Biomater 2009, 5 (8), 3007-3020. (13)Kundu, S. C.; Dash, B. C.; Dash, R.; Kaplan, D. L. Natural Protective Glue Protein, Sericin Bioengineered by Silkworms: Potential for Biomedical and Biotechnological Applications. Progress in Polymer Science 2008, 33 (10), 998-1012. (14)Xie, H.; Yang, W.; Chen, J.; Zhang, J.; Lu, X.; Zhao, X.; Huang, K.; Li, H.; Chang, P.; Wang, Z.; Wang, L. A Silk Sericin/Silicone Nerve Guidance Conduit Promotes Regeneration of a Transected Sciatic Nerve. Adv Healthc Mater 2015, 4 (15), 2195-2205.

28


Page 29 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(15)Akturk, O.; Tezcaner, A.; Bilgili, H.; Deveci, M. S.; Gecit, M. R.; Keskin, D. Evaluation of Sericin/Collagen Membranes as Prospective Wound Dressing Biomaterial. J Biosci Bioeng 2011, 112 (3), 279-288. (16)Siritienthong,

T.;

Ratanavaraporn,

J.;

Aramwit,

P.

Development

of

Ethyl

Alcohol-Precipitated Silk Sericin/Polyvinyl Alcohol Scaffolds for Accelerated Healing of Full-Thickness Wounds. Int J Pharm 2012, 439 (1-2), 175-186. (17)Zhang, Y. Q.; Ma, Y.; Xia, Y. Y.; Shen, W. D.; Mao, J. P.; Xue, R. Y. Silk Sericin-Insulin Bioconjugates: Synthesis, Characterization and Biological Activity. J Control Release 2006, 115 (3), 307-315. (18)Aramwit, P.; Palapinyo, S.; Srichana, T.; Chottanapund, S.; Muangman, P. Silk Sericin Ameliorates Wound Healing and Its Clinical Efficacy in Burn Wounds. Arch Dermatol Res 2013, 305 (7), 585-594. (19)Wang, Z.; Zhang, Y.; Zhang, J.; Huang, L.; Liu, J.; Li, Y.; Zhang, G.; Kundu, S. C.; Wang, L. Exploring Natural Silk Protein Sericin for Regenerative Medicine: an Injectable, Photoluminescent, Cell-Adhesive 3D Hydrogel. Sci Rep 2014, 4, 7064. (20)Dash, B. C.; Mandal, B. B.; Kundu, S. C. Silk Gland Sericin Protein Membranes: Fabrication and Characterization for Potential Biotechnological Applications. J Biotechnol 2009, 144 (4), 321-329. (21)Kundu, B.; Kundu, S. C. Silk Sericin/Polyacrylamide in Situ Forming Hydrogels for Dermal Reconstruction. Biomaterials 2012, 33 (30), 7456-7467. (22)Zhang, Y.-Q.; Tao, M.-L.; Shen, W.-D.; Zhou, Y.-Z.; Ding, Y.; Ma, Y.; Zhou, W.-L.

29



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Immobilization of l-Asparaginase on the Microparticles of the Natural Silk Sericin Protein and Its Characters. Biomaterials 2004, 25 (17), 3751-3759. (23)Cho, K. Y.; Moon, J. Y.; Lee, Y. W.; Lee, K. G.; Yeo, J. H.; Kweon, H. Y.; Kim, K. H.; Cho, C. S. Preparation of Self-Assembled Silk Sericin Nanoparticles. Int J Biol Macromol 2003, 32 (1-2), 36-42. (24)Mandal, B. B.; Kundu, S. C., Self-Assembled Silk Sericin/Poloxamer Nanoparticles as Nanocarriers of Hydrophobic and Hydrophilic Drugs for Targeted Delivery. Nanotechnology 2009, 20 (35), 355101. (25)Das, S. K.; Dey, T.; Kundu, S. C. Fabrication of Sericin Nanoparticles for Controlled Gene Delivery. RSC Advances 2014, 4 (5), 2137. (26)Z.L. Tyrrell, Y. S., M. Radosz Fabrication of Micellar Nanoparticles for Drug Delivery Through the Self-Assembly of Block Copolymer. Progress in Polymer Science 2010, 35 (9), 1128-1143. (27) Lu, Y.; Low, P. S. Folate-Mediated Delivery of Macromolecular Anticancer Therapeutic Agents. Adv Drug Deliv Rev 2002, 54 (5), 675-693. (28)Gu, Y. J.; Cheng, J.; Jin, J.; Cheng, S. H.; Wong, W. T. Development and Evaluation of pH-Responsive Single-Walled Carbon Nanotube-Doxorubicin Complexes in Cancer Cells. Int J Nanomedicine 2011, 6, 2889-2898. (29)Li,

D.;

Tang,

J.;

Wei,

C.;

Guo,

J.;

Wang,

S.;

Chaudhary,

D.;

Wang,

C.

Doxorubicin-Conjugated Mesoporous Magnetic Colloidal Nanocrystal Clusters Stabilized by Polysaccharide as a Smart Anticancer Drug Vehicle. Small 2012, 8 (17), 2690-2697.

30


Page 31 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30)Takasu, Y.; Yamada, H.; Tsubouchi, K. Isolation of Three Main Sericin Components From the Cocoon of the Silkworm, Bombyx mori. Biosci Biotechnol Biochem 2002, 66 (12), 2715-2718. (31)Bruckman, M. A.; Randolph, L. N.; VanMeter, A.; Hern, S.; Shoffstall, A. J.; Taurog, R. E.; Steinmetz, N. F. Biodistribution, Pharmacokinetics, and Blood Compatibility of Native and PEGylated Tobacco Mosaic Virus Nano-Rods and -Spheres in Mice. Virology 2014, 449, 163-173. (32)Banerjee, S. S.; Chen, D. H. Multifunctional pH-Sensitive Magnetic Nanoparticles for Simultaneous Imaging, Sensing and Targeted Intracellular Anticancer Drug Delivery. Nanotechnology 2008, 19 (50), 505104. (33)El-Gogary, R. I.; Rubio, N.; Wang, J. T.; Al-Jamal, W. T.; Bourgognon, M.; Kafa, H.; Naeem, M.; Klippstein, R.; Abbate, V.; Leroux, F.; Bals, S.; Van Tendeloo, G.; Kamel, A. O.; Awad, G. A.; Mortada, N. D.; Al-Jamal, K. T. Polyethylene Glycol Conjugated Polymeric Nanocapsules for Targeted Delivery of Quercetin to Folate-Expressing Cancer Cells in Vitro and in Vivo. ACS Nano 2014, 8 (2), 1384-1401. (34) Cho, Y. W.; Park, S. A.; Han, T. H.; Son, D. H.; Park, J. S.; Oh, S. J.; Moon, D. H.; Cho, K.-J.; Ahn, C.-H.; Byun, Y.; Kim, I.-S.; Kwon, I. C.; Kim, S. Y. In Vivo Tumor Targeting and Radionuclide Imaging with Self-Assembled Nanoparticles: Mechanisms, Key Factors, and Their Implications. Biomaterials 2007, 28 (6), 1236-1247. (35)Owens, D. E., 3rd; Peppas, N. A. Opsonization, Biodistribution, and Pharmacokinetics of Polymeric Nanoparticles. Int J Pharm 2006, 307 (1), 93-102.

31



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(36)Alexis, F.; Pridgen, E.; Molnar, L. K.; Farokhzad, O. C. Factors Affecting the Clearance and Biodistribution of Polymeric Nanoparticles. Mol Pharm 2008, 5 (4), 505-515. (37)Xiao, K.; Li, Y.; Luo, J.; Lee, J. S.; Xiao, W.; Gonik, A. M.; Agarwal, R. G.; Lam, K. S. The Effect of Surface Charge on in Vivo Biodistribution of PEG-Oligocholic Acid Based Micellar Nanoparticles. Biomaterials 2011, 32 (13), 3435-3446. (38)Lundqvist, M.; Stigler, J.; Elia, G.; Lynch, I.; Cedervall, T.; Dawson, K. A. Nanoparticle Size and Surface Properties Determine the Protein Corona with Possible Implications for Biological Impacts. Proc Natl Acad Sci U S A 2008, 105 (38), 14265-14270. (39)Ai, J.; Biazar, E.; Jafarpour, M.; Montazeri, M.; Majdi, A.; Aminifard, S.; Zafari, M.; Akbari, H. R.; Rad, H. G. Nanotoxicology and Nanoparticle Safety in Biomedical Designs. Int J Nanomedicine 2011, 6, 1117-1127. (40)Jiang, T.; Li, Y. M.; Lv, Y.; Cheng, Y. J.; He, F.; Zhuo, R. X., Amphiphilic Polycarbonate Conjugates of Doxorubicin With pH-Sensitive Hydrazone Linker for Controlled Release. Colloids Surf B Biointerfaces 2013, 111C, 542-548. (41)Nakanishi, T.; Fukushima, S.; Okamoto, K.; Suzuki, M.; Matsumura, Y.; Yokoyama, M.; Okano, T.; Sakurai, Y.; Kataoka, K. Development of the Polymer Micelle Carrier System for Doxorubicin. J Control Release 2001, 74 (1-3), 295-302. (42)Vasir, J. K.; Labhasetwar, V. Biodegradable Nanoparticles for Cytosolic Delivery of Therapeutics. Adv Drug Deliv Rev 2007, 59 (8), 718-728. (43)Ren, D.; Kratz, F.; Wang, S. W., Protein Nanocapsules Containing Doxorubicin as a pH-Responsive Delivery System. Small 2011, 7 (8), 1051-1060.

32


Page 33 of 34

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(44)Yang, X.; Grailer, J. J.; Rowland, I. J.; Javadi, A.; Hurley, S. A.; Matson, V. Z.; Steeber, D. A.; Gong, S. Multifunctional Stable and pH-Responsive polPmer Vesicles Formed by Heterofunctional Triblock Copolymer for Targeted Anticancer Drug Delivery and Ultrasensitive MR Imaging. ACS Nano 2010, 4 (11), 6805-6817. (45)Chen, W. H.; Lei, Q.; Luo, G. F.; Jia, H. Z.; Hong, S.; Liu, Y. X.; Cheng, Y. J.; Zhang, X. Z. Rational Design of Multifunctional Gold Nanoparticles via Host-Guest Interaction for Cancer-Targeted Therapy. ACS Appl Mater Interfaces 2015, 7 (31), 17171-17180. (46)Meng, H.; Xue, M.; Xia, T.; Zhao, Y. L.; Tamanoi, F.; Stoddart, J. F.; Zink, J. I.; Nel, A. E. Autonomous in Vitro Anticancer Drug Release From Mesoporous Silica Nanoparticles by pH-Sensitive Nanovalves. J Am Chem Soc 2010, 132 (36), 12690-12697. (47)Yoshimori, T.; Yamamoto, A.; Moriyama, Y.; Futai, M.; Tashiro, Y. Bafilomycin A1, a Specific Inhibitor of Vacuolar-Type H(+)-ATPase, Inhibits Acidification and Protein Degradation in Lysosomes of Cultured Cells. J Biol Chem 1991, 266 (26), 17707-17712.

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Design and Fabrication of Multifunctional Sericin ... - ACS Publications

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