Double-Cross-Linked Hyaluronic Acid Nanoparticles with pH

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Double-crosslinked hyaluronic acid nanoparticles with pH/reduction dual-responsive triggered release and pH-modulated fluorescence for folate receptor-mediated targeting visualized chemotherapy Xubo Zhao, Xu Jia, Lei Liu, Jin Zeng, Kun Tian, Tingting Zhou, and Peng Liu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b00102 • Publication Date (Web): 24 Mar 2016 Downloaded from http://pubs.acs.org on March 24, 2016

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Double-crosslinked hyaluronic acid nanoparticles with pH/reduction dual-responsive triggered release and pH-modulated fluorescence for folate receptormediated targeting visualized chemotherapy Xubo Zhao, Xu Jia, Lei Liu, Jin Zeng, Kun Tian, Tingting Zhou and Peng Liu* State Key Laboratory of Applied Organic Chemistry and Key Laboratory of Nonferrous Metal Chemistry and Resources Utilization of Gansu Province, College of Chemistry and Chemical Engineering, Lanzhou University, Lanzhou 730000, China ABSTRACT: A versatile folate receptor-mediated targeting tumor theranostics has been designed for pH/reduction dual-responsive controlled anticancer drug release and pH-modulated fluorescent tumor imaging via facile ionic (pH sensitive) and covalent (reduction responsive) double-crosslinking (DCL) of the folic acid (FA) and Rhodamine 6G modified hyaluronic acid (FA-HA-Rh 6G). After optimizing the morphology and diameter of the resultant nanoparticles (DCL FA-HA-Rh 6G NPs) via modulating the concentration of the ionic and covalent crosslinking agents, the one with Ca and S contents of 1.70 wt % and 2.84 wt % and an average hydrodynamic diameter of 154 nm was chosen as the desired DDS for DOX. They not only had high drug loading capacity (DLC) and drug encapsulation efficiency (DEE) (716±34 mg/g and 71.6±3.4%), but also possessed perfect triggered release and strong fluorescence intensity in the

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stimulated tumor microenvironment. The MTT assay and CLSM analysis revealed that the proposed double-crosslinked HA-based DDS had favorable cytocompatibility and folate receptor-mediated targeting functionality to the HeLa cells, and could obviously enhance the anticancer efficiency of DOX. The integration of the pH and reduction dual-responsiveness, folate receptor-mediated targeting functionality and pH-dependent fluorescence intensity into the biodegradable and biocompatible HA nanoparticles make the DCL FA-HA-Rh 6G NPs significant potential for future visualized chemotherapy of cancers.

Keywords: drug delivery system; ionic and covalent double crosslinking; folate receptormediated targeting; pH/reduction triggered release; pH-modulated fluorescence

INTRODUCTION In last decades, smart drug delivery system (DDS) have been intensely developed for therapy of various diseases, such as Alzheimer's disease,1 epilepsy,2 human immunodeficiency virus,3 malaria,4 rheumatoid arthritis,5 diabetes mellitus6 and especially cancer,7-9 due to their unique structure and outstanding performance. Especially for cancers, to efficiently improve the chemotherapy efficiency and avoid the severe side effects in healthy tissues, controlled release of anticancer drugs has attracted extensive attention, with the help of multifunctional DDS,10,11 such as micelles,12,13 graphene,14,15 mesoporous silica nanoparticle (MSN),16 synthetic or natural polymers-based nanocarriers,17,18 and so on. However, the artificial engineered DDS still face serious challenges by now, i.e. regulated release of anticancer drug, biodegradability, and tumortargeted delivery.19


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It is fortunate that the physicochemical microenvironment differences between tumor and normal tissues could be utilized to regulate the release of anticancer drugs. In contrast to normal tissues, the physicochemical features of tumor tissues include weak acidity,20 overexpressed proteins and enzymes21 and abnormal temperature gradients.22 Most importantly, the endosomes and lysosomes of tumor tissues are reductive state due to high level of glutathione (GSH) or cysteine.23 Based on these apparently differences abovementioned, anticancer drugs could be targeted to the desired targeting sites.24-27 Additionally, the tumor cells or tissues usually overexpress certain cell surface receptors, which could also be utilized to make DDS targeting to specific cells or tissues.18,28,29 Among them, folate receptors (FRs) have been widely employed to modify the DDS, so they could be selectively bound onto the targeted cells and internalized via FR-mediated endocytosis.30-32 Most recently, tumor theranostics integrating therapeutic and diagnostic functions into “all-inone” multi-functional biomedical platform has shown great advantage for convenient real-time tracking the therapeutic effects during the chemotherapy treatment course.33 Especially, the pHmodulated fluorescence fluorochrome, which experience the fluorescence intensity reversibly switches with the change of media pH,34 could be used for the fluorescent tumor imaging. For example, Rhodamine 6G (Rh 6G) and its derivatives, which possess pH-dependent fluorescence by activating carbonyl group in spirolactone in acidic media, exhibit strong fluorescence intensity at acidic media (tumor microenvironment), while their fluorescence intensity is very weak at neutral and basic media (physiological medium). Thus, it is a promising multifunctional platform for visualized chemotherapy by introducing such tumor-microenvironment sensitive imaging functional groups into the DDS.35 The natural polysaccharides, such as alginate,36 chitosan,37 and hyaluronic acid (HA),38 have been widely used for DDS, due to their favorable biocompatibility and biodegradable property.


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Among them, HA has attracted extensive attention because of its specific binding with the CD44 hyaluronic acid receptor, which is also overexpressed in some tumor cells.39 HA has been crosslinked with either disulfide crosslinker40 or Ca2+

41

to form the nanogels for biomedical

application, with the single reduction or pH stimuli-responsive property. By now, there is no report on the HA-based DDS crosslinked with both ionic and covalent bonds. Obviously, the ionic and covalent double-crosslinking possesses favorable pH and reduction dual-stimuli sensitive properties, which is favorable to modulate drug release in tumor tissues triggered by their acidic and reductive condition. In the present work, a novel double-crosslinked pH and reduction dual-responsive HA-based DDS (DCL FA-HA-Rh 6G NPs) with targeting functionality and pH-dependent fluorescence intensity has been developed via the facile ionic and covalent double-crosslinking method for the first time. The influences of the concentration of the ionic and covalent crosslinkers on the morphologies and diameters of the DCL FA-HA-Rh 6G NPs were optimized in detail. The integration of the pH and reduction dual-responsive, FR-mediated targeting functionality and pHdependent fluorescence intensity into the biodegradable and biocompatible HA nanoparticles make the DCL FA-HA-Rh 6G NPs significant potential for future visualized chemotherapy of cancers.

EXPERIMENTAL SECTION Materials and Reagents. Sodium hyaluronate (MW = 400 000 Da) was purchased from Shandong Freda Biopharm. Co., Ltd. Doxorubicin hydrochloride (DOX) was obtained from Beijing Huafeng United Technology Co., Ltd. 1-Ethyl-3-(3-dimethyl aminopropyl) carbodiimide hydrochloride (EDCl) was purchased from Fluorochem. N-Hydroxylsuccinimide (NHS) was


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supplied by Aladdin Chemistry Co., Ltd. Cystamine dihydrochloride (Cy) was purchased from J & K Chemical Ltd. Glutathione was provided by Tianjin Heowns Biochem. Co., Ltd. Folic acid (FA) and other reagents were analytical reagent grade, purchased from Tianjin Chemical Company, China. Deionized water was used throughout.

Synthesis of DCL FA-HA-Rh 6G NPs. Rh 6G-NH2 was synthesized according to the procedure reported previously:42 ethanediamine (0.242 g, 4.0 mmol) was added into a solution of Rh 6G (0.961 g, 2.0 mmol) in EtOH (20 mL). The solution was refluxed under N2 atmosphere for 24 h and then condensed in vacuum. Finally the product, Rh 6G-NH2, was obtained with a yield of 40%, after purified by flash column chromatography (petroleum/CH2Cl2 = 2:1 to 1:1). The amidation of FA was carried out according to the published literature:43 after FA (0.441 g, 1.0 mmol) was activated with DCC (0.248 g, 1.2 mmol) and NHS (0.230 g, 2.0 mmol) in 30 mL dimethyl sulfoxide (DMSO) at 50 oC for 6 h, it was then reacted with ethylenediamine (10 mmol) with pyridine (15 µL) as catalyst at room temperature overnight. N, N'-Dicyclohexylurea (DCU) was removed by filtration, and the crude product was then precipitated by addition of eight-fold volume of acetonitrile, filtered, and washed three times with diethyl ether, and dried in vacuum finally. 0.403 g HA (1.0 mmol), 0.230 g NHS (1.9 mmol), 0.023 g DCC (0.12 mmol), and 50 µL pyridine were added to 15 mL DMSO with magnetic stirring for 30 min. Subsequently, 0.013 g Rh 6G-NH2 (0.03 mmol) and 0.134 g FA-NH2 (0.32 mmol) were introduced into the above mixture under vigorously stirring at 50 oC for 24 h. Insoluble substance containing DCU was removed by vacuum filtration. Subsequently, the solution was transferred into dialysis tubes (MWCO of 1000) and dialyzed extensively against DMSO for 24 h to remove the unreacted FA-


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NH2 and Rh 6G-NH2, followed by dialysis against water for 72 h to remove DMSO. Finally, the resulting FA-HA-Rh 6G was obtained by lyophilization. The calcium ion (Ca2+) and cystamine (Cy) were utilized as pH-responsive and reductionresponsive crosslinker respectively to fabricate the pH/reduction dual-responsive HA-based microgel via electrostatic interaction between Ca2+ and carboxyl groups of HA, and amidation reaction between the amino groups of Cy and carboxyl groups of HA, respectively. Typically, 50 mg FA-HA-Rh 6G was dissolved in 50 mL water. 3.01 g CaCl2 (0.027 mol) pre-dissolved in 30 mL water was added to the FA-HA-Rh 6G solution dropwise with vigorous stirring at room temperature for 24 h. The ionic-crosslinked NPs (ICL FA-HA-Rh 6G NPs3) were obtained via centrifugation and washing three times with water to remove free Ca2+ afterwards. Finally, the dispersion of the ICL FA-HA-Rh 6G NPs was added into ethanol dropwise to solidify their morphology. 40 mg ICL FA-HA-Rh 6G NPs was dispersed into 40 mL water. 0.006 g cystamine dihydrochloride (0.027 mmol), 0.014 g NHS (0.12 mmol), 0.019 g EDCI (0.16 mmol) and 25 µL triethylamine were introduced into the above solution for the amidation reaction with magnetic stirring at normal temperature. The product was obtained by centrifugation. After dialysis against water for 48 h, the product, double-crosslinked NPs (DCL FA-HA-Rh 6G NPs), was obtained by adding its aqueous dispersion into ethanol by dropwise followed by freeze-drying.

Drug loading and triggered release. The DCL FA-HA-Rh 6G NPs3 (10.0 mg) were dispersed into 10.0 mL 1.0 mg/mL DOX aqueous solution for drug-loading with the aid of ultrasound, and then the dispersion was adjusted to pH 5.0. After being magnetically stirred until uniform and swung by a table concentrator for 24 h in dark, the DOX-loaded DCL FA-HA-Rh 6G NPs3 were centrifuged to remove the excess DOX. The drug concentration in the supernatant solution was


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monitored using a UV-vis spectrophotometer at 480 nm to assess the drug-loading capacity (DLC) and the drug encapsulation efficiency (DEE). Sequentially, the DOX-loaded DCL FA-HA-Rh 6G NPs3 were dispersed into 10 mL PBS mimicking different physiological media respectively, transferred into the dialysis tube (molecular weight cutoff of 12 000), and immersed into 120 mL relevant PBS at 37 °C. Each aliquot of 5.0 mL was taken out at certain intervals, and the drug concentration was measured with UV-vis spectrophotometer to assess the cumulative release of DOX. After each sampling, 5.0 mL fresh relevant PBS was added to keep the total volume constant.

Cell toxicity and celluler uptake. MTT assay was performed to evaluate the cytocompatibility of the DCL FA-HA-Rh 6G NPs3 and DOX-loaded DCL FA-HA-Rh 6G NPs3 in HeLa cells. In addition, the inhibition growth efficiency and FR-mediated targeting property of the DOX-loaded DCL FA-HA-Rh 6G NPs3 were evaluated in the absence or presence of 10 mM GSH-OEt and 1 mM free FA, in comparison with that incubated with free DOX. For the MTT assay, the cells were seeded into 96-well plates at densities of 1×105 cells per well. After the incubation with different concentrations of the samples for 24 h, the cells were washed with PBS and processed for MTT assay to determine the cell viability. The intracellular release of DOX from the DOX-loaded DCL FA-HA-Rh 6G NPs3 in the absence or presence of 1 mM free FA were determined by confocal laser scanning microscopy (CLSM) (LYMPUS FV-1000) in HeLa cells after 12 h of incubation, with excitation wavelengths of 405 nm for Hoechst and 480 nm for DOX.


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Instruments and characterization. Elemental analysis of the DCL FA-HA-Rh 6G NPs3 was performed on an Elementar vario EL instrument (Elementar Analysensysteme GmbH, Munich, Germany). Thermogravimetric analysis (TGA) was conducted with a TA Instrument 2050 thermogravimetric analyzer at 10 °C/min from 25 to 800 °C at nitrogen atmosphere. A Bruker IFS 66 v/s infrared (IR) spectrometer (Bruker, Karlsruhe, Germany) was used for the FT-IR analysis in the range of 400-4000 cm-1 with a resolution of 4 cm-1. The zeta potentials of the nanoparticles were determined using a Zetasizer Nano ZS (Malvern Instruments Ltd., UK). The morphology of the FA-HA-Rh 6G NPs3 was characterized using a JEM1200 EX/S transmission electron microscope (TEM). The mean hydrodynamic diameter and distribution, ionic strength and reduction-responsive properties, and stability of the NPs were determined using the dynamical mode [dynamic light scattering (DLS)] on a Light Scattering System BI-200SM device (Brookhaven Instruments) equipped with a BI-200SM goniometer, a BI-9000AT correlator, a temperature controller, and a coherent INOVA 70C argon ion laser at 20 °C. DLS measurements are performed using a 135mW intense laser excitation at 514.5 nm at a detection angle of 90° using their dispersions (0.5 mg/mL) at 25 °C. Fluorescence

spectra

were

obtained

with

a

PerkinElmer

LS

55

luminescence

spectrophotometer equipped with a 1.0 cm quartz cell. The pH responsive fluorescence spectrum was conducted in the wavelength range of 500-650 nm upon excitation at 525 nm. Typically, 1.0 mL of a 10 µg/mL DCL FA-HA-Rh 6G NPs3 dispersions with different pH values were added into the color comparison tubes, and the synchronous fluorescence spectra were recorded in 480680 nm with ∆λ of 50 nm.


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Scheme 1. Schematic diagram of the synthesis of FA-HA-Rh 6G.

RESULTS AND DISCUSSION Synthesis and characterization of DCL FA-HA-Rh 6G NPs. In order to render the HA-based DDS both targeting and fluorescent imaging properties, FA and Rh 6G moieties were decorated onto the skeleton of HA (Scheme 1). The product became dark red in comparison with the original white one (insert in Fig. 1A). The appearance of new absorbance at 280 nm of FA and 560 nm of Rh 6G also demonstrated that FA and Rh 6G segments had been decorated onto the skeleton of HA (Fig. 1A), with the degree of substitution (DS) of 12.5% and 0.64% for FA and Rh 6G respectively, calculated from their calibration curves.44,45


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1.4 1.2 1.0 0.8 0.6 0.4 0.2

HA FA-HA-Rh 6G DCL FA-HA-Rh 6G NPs3

0.0

300

400

500

600

Wavelength (nm)

B Transmittance (%)

Absorption

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1631 1716

1363 1223

1375 1355

FA-NH2 Rh 6G-NH2

1230

HA FA-HA-Rh 6G

1800

1500

1200

FA

900

600

-1

Wavenumber(cm

)


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100

C

FA-HA-Rh 6G DCL FA-HA-Rh 6G NPs3

Weight (%)

80

60

40

20 100

200

300

400

500

600

Temperature ( C) o

500

Fluorescence intensity (a.u.)

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pH 4.0 pH 5.0 pH 6.0 pH 7.0 pH 8.0

400 300 200 100 0 525

550

575

600

625

650

675

700

Emission wavelength (nm) Fig. 1. A) UV-vis spectra of HA, FA-HA-Rh 6G and DCL FA-HA-Rh 6G NPs3; B) FT-IR spectra

of HA, FA, FA-NH2, Rh 6G-NH2, and FA-HA-Rh 6G; C) TGA curves of FA-HA-Rh 6G and DCL FA-HA-Rh 6G NPs3; D) Fluorescent spectra of DCL FA-HA-Rh 6G NPs3 (10 µg/mL) suspended in deionized water with pH rising from 4.0 to 8.0. The inset is the image of DCL FAHA-Rh 6G NPs3 in neutral (pH 7.0) (a) and acid (pH 5.0) (b) solution under 365 nm-UV light.


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Fig. 2. TEM images of ICL FA-HA-Rh 6G NPs1 (A), ICL FA-HA-Rh 6G NPs2 (B), ICL FA-HA-

Rh 6G NPs3 (C), DCL FA-HA-Rh 6G NPs1 (D), DCL FA-HA-Rh 6G NPs2 (E), DCL FA-HA-Rh 6G NPs3 (F), DCL FA-HA-Rh 6G NPs3 treated with 10 mM GSH (G), DCL FA-HA-Rh 6G NPs3 in pH 5.0 for 8 h (H), and DCL FA-HA-Rh 6G NPs3 treated with 10 mM GSH and pH 5.0 for 8 h (I).


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In the FT-IR spectrum of the FA-HA-Rh 6G (Fig. 1B), the characteristic absorbance at 1631 cm−1 of carbonyl stretching vibration in amide group revealed that the carboxyl groups of HA had been successfully amidated. The characteristic absorbance of FA at 1230 cm-1 shifted to 1237 cm-1 in the product, as reported previously.46 And the characteristic absorbance peaks of Rh 6G at 1355 and 1375 cm-1 appeared at 1363 cm-1 in the product.47 These absorbance peaks demonstrated the successful conjugation of HA with FA-NH2 and Rh 6G-NH2. Meantime, the characteristic absorbance at 1716 cm−1 still could be observed in the FT-IR spectrum, assigning to the carboxyl groups both retained in HA and introduced by FA-NH2. Then the ionic-crosslinked NPs (ICL FA-HA-Rh 6G NPs) were synthesized by crosslinking the FA-HA-Rh 6G with Ca2+. The ICL FA-HA-Rh 6G NPs exhibited morphology from irregular sphere to well-define spherical shape with increasing the concentration of Ca2+ (Fig. 2). And their diameters increased from 42±6, 52±4, to 66±8 nm with increasing the concentration of CaCl2 from 0.067, 0.100, to 0.133 g mL-1 (with molar ratio between Ca2+ and carboxyl acid group in FA-HA-Rh 6G of 150, 225 and 300), respectively. All of the NPs showed obvious aggregates and broad particle size distribution. The corona could be seen in the TEM images, especially in that of the ICL FA-HA-Rh 6G NPs3 (Fig. 2C). This phenomenon might be resulted from the uncrosslinked FA-HA-Rh 6G segments on their surface, which extended out as the corona. And the extended FA-HA-Rh 6G segments interacted each other with those on other nanoparticles, causing the aggregation. Finally, the ICL FA-HA-Rh 6G NPs were further covalently crosslinked with Cy to make the HA-based DDS in response to high level cysteine or GSH in the cytoplasm and endolysosomes. After the crosslinking with Cy, the corona in the ICL FA-HA-Rh 6G NPs disappeared. The double-crosslinked NPs (DCL FA-HA-Rh 6G NPs) showed bigger particle size of 86±6, 77±9 and 109±7 nm, compared with the ICL FA-HA-Rh 6G NPs crosslinked with CaCl2 with the


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molar ratio between Ca2+ and carboxyl acid group in FA-HA-Rh 6G of 150, 225 and 300 respectively. As vividly depicted in Fig. 2, the DCL FA-HA-Rh 6G NPs3 had greater diameter, well-defined construction, and the aggregates had been obviously reduced in comparison with the ICL FA-HA-Rh 6G NPs3, attributing to the crosslinking of the extended FA-HA-Rh 6G segments on their surface during the covalent crosslinking. Furthermore, the zeta potential of the DCL FA-HA-Rh 6G NPs3 after covalent crosslinking with cystamine increased to -38.5 mV, compared to the zeta potential (-44.1 mV) of the ICL FA-HA-Rh 6G NPs3 at pH 7.4. It also demonstrated the consumption of carboxyl groups in the amidation with Cy. Owing to the perfect morphology and unique size of the DCL FA-HA-Rh 6G NPs3, they were chosen as DDS for further investigation. In the TGA analysis (Fig. 1C), the FA-HA-Rh 6G remained about 24.2% of its carbonization product upon 600 °C, whereas the DCL FA-HA-Rh 6G NPs3 gave a residual mass of 27.8%. Assuming the Ca2+ ions in the nanoparticles turned into CaCO3 in the heating procedure, the increased residual mass of the DCL FA-HA-Rh 6G NPs3 should be come from the CaCO3 formed. Therefore, it could be calculated that the ICL FA-HA-Rh 6G NPs3 contained 1.70 wt% Ca2+. It meant that 42% of carboxyl acid groups in HA had been ionic-crosslinked by Ca2+. The Ca2+ content in the DCL FA-HA-Rh 6G NPs3 was much lower than the feeding ratio of Ca2+ in the ionic-crosslinking procedure, because of the relatively lower carboxyl acid group content than others such as alginic acid, high molecular weight, as well as the amidation with the rigid FA and Rh 6G groups. The element analysis showed the sulfur content of the DCL FA-HA-Rh 6G NPs3 was 2.84%, indicating that 43% of carboxyl acid groups in HA had been covalently crosslinked by Cy.


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pH-modulated fluorescent property. The fluorescence intensity of the DCL FA-HA-Rh 6G

NPs3 upon excitation at 570 nm increased with decreasing the media pH value from 8.0 to 4.0, especially from 6.0 to 4.0 (Fig. 1D), due to the spirolactam ring-opening of Rh 6G, in acidic solution by activating the carbonyl group in spirolactone.34 Meanwhile, the solution transformed from colorless to pink in the daylight, and changed from colorless to yellow under the ultraviolet lamp at the wavelength of 365 nm (insert in Fig. 1D), due to that the spiro form turned into the ring-opened amide form as a function of the hydrogen ion when the solution was varied from alkaline to acidic. The strong fluorescence intensity in the acidic media is expected to be used for the real-time fluorescent imaging, so as to track the therapeutic effects during the chemotherapy treatment course.

pH/reduction dual-responsive properties. In the DLS analysis, the DCL FA-HA-Rh 6G NPs3

displayed an average hydrodynamic diameter (Dh) of 154 nm and a narrow size distribution at pH 7.4 (Fig. 3A), consistent with the TEM results. Compared with the ICL FA-HA-Rh 6G NPs3, their Dh became smaller and their size distribution changed narrower (Fig. 3A), also revealing the covalent crosslinking by Cy. The effect of the media pH value on the Dh of the DCL FA-HARh 6G NPs3 was studied with DLS after they were dispersed into PBS with various pH values for 8 h (Fig. 3B). With decreasing the media pH value from 8.0 to 4.0, their Dh increasd from 124 nm to 208 nm. The acidic media probably weakens the interaction between Ca2+ and HA. At acidic media, HA deionizes and part of the Ca2+-crosslinked carboxylate groups protonates into carboxylic acid groups, unfolding the corresponding Ca2+-crosslinking linkages. Decreasing pH value even to 4.0, the DCL FA-HA-Rh 6G NPs3 did not disintegrate. It could still keep welldefined spherical shape in pH 5.0 media, revealing the single covalent crosslinking could solidify the structure of the HA-based DDS.48


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Owing to the instability of the crosslinking structure in slightly acidic and reductive environment, excessive GSH (10 mM) and acidic medium (pH 5.0) were introduced to evaluate the dissociation of the DCL FA-HA-Rh 6G NPs3. After stirring at room temperature for 8 h, massive smaller matters were found (Fig. 3A), implying the DCL FA-HA-Rh 6G NPs3 were decrosslinked successfully in the acidic reductive media, also consistent with TEM analysis (Fig. 2I).

To investigate the disassembly of the DCL FA-HA-Rh 6G NPs3 under different pH and reductant levels, the stability of the ICL FA-HA-Rh 6G NPs3 was tracked under different acidic media (pH 7.0, pH 6.0, and pH 5.0) using DLS analysis. With the decreasing of pH of dispersion media from pH 7.0 to pH 5.0, the typical hydrodynamic diameter distribution of the ICL FA-HARh 6G NPs3 became broader, and the diameters changed larger (Fig. 3C). It was resulted from the unfolding the corresponding Ca2+-crosslinking linkages. Meanwhile, abundant small matters were observed in DLS analysis at pH 6.0 and pH 5.0. The polydispersity index of the ICL FAHA-Rh 6G NPs3 also kept in consistent with the average hydrodynamic diameter distribution at varying pH values. Taken the above results into consideration, pH 5.0 was chosen as a basic condition to investigate the effect of the GSH concentration on the stability of the DCL FA-HA-Rh 6G NPs3. Increasing the GSH concentration from 0, to 0.2 µM, 10 µM, and 10 mM, the the hydrodynamic diameter distribution of the DCL FA-HA-Rh 6G NPs3 became broader, and the diameters changed larger (Fig. 3D), similar as the ICL FA-HA-Rh 6G NPs3 in pH 5.0 medium. However, the small matters could only be observed in DLS analysis at pH 5.0 with higher GSH concentrations (10 µM, and 10 mM GSH). In particular, at the medium of pH 5.0 with 10 mM GSH, the greatly excess of GSH concentration and slightly acidic condition led to the dissociation of the DCL FA-HA-Rh 6G NPs3, by unfolding both the ionic and covalent


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crosslinking linkages. On the basis of above analysis, it could be concluded that the DCL FAHA-Rh 6G NPs3 had excellent stability at low concentration of GSH and neutral conditions, while dissociated at acidic conditions with high concentration of GSH (pH 5.0 with 10 mM GSH). This feature could be used for the targeted delivery of anticancer drug through the trmour microenvironment responsive controlled release. Furthermore, the stability of the DCL FA-HA-Rh 6G NPs3 in various times at PBS (pH 7.4) was also tracked with DLS. As shown in Fig. 3E, the DCL FA-HA-Rh 6G NPs3 remained similar average hydrodynamic diameter and diameter distribution in various times. This phenomenon indicated that the ionically and covalently double-crosslinked FA-HA-Rh 6G NPs3 had stable structure in PBS (pH 7.4) for 24 h at least.

160 140

A

ICL FA-HA-Rh 6G NPs3 DCL FA-HA-Rh 6G NPs3 DCL FA-HA-Rh 6G NPs3 at pH 5.0

120

f(Dh)(%)

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

Biomacromolecules

Disassembled DCL FA-HA-Rh 6G NPs3

100

PDI 0.005

PDI 0.085

80

PDI 0.091

PDI 0.005

60 40 20 0

0

40

80

120

160

200

240

Dh(nm)


17

Biomacromolecules

100

B

PDI 0.078

PDI 0.005 PDI 0.005

pH 8.0 pH 7.0 pH 6.0 pH 5.0 pH 4.0

f(Dh)(%)

80 PDI 0.082

60

PDI 0.005

40 20 0

80

100

120

140

160

180

200

220

240

Dh(nm)

100

C

pH 7.0

PDI 0.005

pH 6.0 pH 5.0

80

f(Dh)(%)

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

Page 18 of 32

60

PDI 0.173 40

PDI 0.242

20 0 50

100

150

200

250

Dh(nm)


18

Page 19 of 32

100

D

No GSH 0.2 µM GSH 10 µ M GSH 10 mM GSH

PDI 0.005

f(Dh)(%)

80

PDI 0.079 60 40

PDI 0.217

PDI 0.005

20 0 0

40

80

120

160

200

240

280

320

Dh(nm)

100

E

80

f(Dh)(%)

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

Biomacromolecules

60

3 h PDI 0.005 6 h PDI 0.005 12 h PDI 0.062 24 h PDI 0.093

40 20 0 100

120

140

160

180

200

Dh(nm)

Fig. 3. The typical hydrodynamic diameter distributions of (A) the ICL FA-HA-Rh 6G NPs3,

DCL FA-HA-Rh 6G NPs3 in water, DCL FA-HA-Rh 6G NPs3 under acidic medium (at pH 5.0), and the disassembly of the DCL FA-HA-Rh 6G NPs3 under excessive GSH (10 mM) and acidic medium (at pH 5.0); (B) the DCL FA-HA-Rh 6G NPs3 in different pH media; (C) the ICL FAHA-Rh 6G NPs3 under different acidic media; (D) the DCL FA-HA-Rh 6G NPs3 under pH 5.0 with different GSH concentrations; and (E) the stability of the DCL FA-HA-Rh 6G NPs3 at pH


19

Biomacromolecules

7.4 PBS for different times.

Drug loading and controlled releasing performance. Owing to the plentiful carboxyl groups in

the DCL FA-HA-Rh 6G NPs3, DOX was chosen as a model drug to assess its drug loading capacity (DLC) and drug encapsulation efficiency (DEE). The DLC and DEE were high as 716±34 mg/g and 71.6±3.4% respectively, via the electrostatic interaction between the carboxyl group of HA and amino group of DOX.18 These data were higher than that of the bi-component CS/SAL NPs,49 due to its single polyanion component. Additionally, the zeta potential increased from -38.5 to -24.2 mV after the DOX-loading, also revealing the electrostatic interaction between the drug and carrier.

Cumulative release of DOX (%)

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

Page 20 of 32

70

A

60

pH 7.4 no GSH pH 7.4 10 µM GSH pH 6.5 no GSH

50

pH 5.0 no GSH pH 5.0 10 mM GSH

40 30 20 10 0 0

200 400 600 800 1000 1200 1400 1600 1800

Time (min)


20

Page 21 of 32

-22

a

B

70

-24

zeta potential (mV)

60 -26

50

-28

40

-30

30 20

-32

10

b

-34

Cumulative release of DOX (%)

0 0

400

800

1200

1600

Time (min) Concentrationof DCL FA-HA-Rh 6G NPs3 (µg/mL) 0 50 100 150 200 250 300 100

C

f e

Cell Viability (%)

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

Biomacromolecules

cd

80 60

ab

40 20 0

3

9 6 DOX equivalent dose (µg/mL)

18

Fig. 4. A) Cumulative DOX release from the DOX-loaded DCL FA-HA-Rh 6G NPs3 in

simulated body fluids. Results show UV-vis analysis at room temperature at 480 nm for DOX. Each value represents the mean (SD

Double-Cross-Linked Hyaluronic Acid Nanoparticles with pH

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