pH-Sensitive Coiled-Coil Peptide-Cross-Linked Hyaluronic Acid

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pH-Sensitive Coiled-Coil Peptide-Crosslinked Hyaluronic Acid Nanogels: Synthesis and Targeted Intracellular Protein Delivery to CD44 Positive Cancer Cells Lingling Ding, Yu Jiang, Jian Zhang, Harm-Anton Klok, and Zhiyuan Zhong Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b01664 • Publication Date (Web): 29 Dec 2017 Downloaded from http://pubs.acs.org on December 29, 2017

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Biomacromolecules

pH-Sensitive Coiled-Coil Peptide-Crosslinked Hyaluronic Acid Nanogels: Synthesis and Targeted Intracellular Protein Delivery to CD44 Positive Cancer Cells

Lingling Ding,† Yu Jiang, † Jian Zhang, †,* Harm-Anton Klok, †,‡ Zhiyuan Zhong †,*

†

Biomedical Polymers Laboratory, and Jiangsu Key Laboratory of Advanced Functional

Polymer Design and Application, College of Chemistry, Chemical Engineering and Materials Science, Soochow University, Suzhou, 215123, P. R. China.

‡

Laboratoire des Polymères, Institut des Matériaux and Institut des Sciences et Ingénierie

Chimiques, École Polytechnique Fédérale de Lausanne (EPFL), Bâtiment MXD, Station 12, CH-1015 Lausanne, Switzerland

ABCTRACT The clinical translation of protein drugs that act intracellularly is limited by the absence of safe and efficient intracellular protein delivery vehicles. Here, pH-sensitive coiled-coil peptide-crosslinked hyaluronic acid nanogels (HA-cNGs) were designed and investigated for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells. HA-cNGs were obtained with a small size of 176 nm from an equivalent mixture of hyaluronic acid conjugates with GY(EIAALEK)3GC (E3) and GY(KIAALKE)3GC (K3) peptides, respectively, at pH 7.4 by nanoprecipitation. Circular dichroism (CD) proved the formation of coiled-coil structures between E3 and K3 peptides at pH 7.4 while fast uncoiling at pH 5.0. HA-cNGs showed facile loading of cytochrome C (CC) and greatly accelerated CC 1 ACS Paragon Plus Environment

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release under mild acidic conditions (18.4%, 76.8% and 91.4% protein release in 24 h at pH 7.4, 6.0 and 5.0, respectively). Confocal microscopy and flow cytometry displayed efficient internalization of CC-loaded HA-cNGs and effective endosomal escape of CC in MCF-7 cancer cells. Remarkably, HA-cNGs loaded with saporin, a ribosome inactivating protein, exhibited significantly enhanced apoptotic activity to MCF-7 cells with a low IC50 of 12.2 nM. These coiled-coil peptide-crosslinked hyaluronic acid nanogels have appeared as a simple and multifunctional platform for efficient intracellular protein delivery.

Keywords: Coiled-coil; Hyaluronic acid; Nanogels; Protein delivery; Cancer therapy.

1. INTRODUCTION In spite of their high specificity and activity, the clinical translation of anticancer proteins that act intracellularly, such as granzyme B, saporin and cytochrome C (CC), is greatly limited owing to their poor plasma stability, low cell selectivity and uptake, as well as inefficient intracellular trafficking.1-4 In recent years, different nanovehicles such as nanocapsules,5,6 polyion nanocomplexes,7,8 polymersomes,9 and hydrogels/nanogels10-12 have been developed for enhanced intracellular protein delivery in vitro and in vivo. In particular, nanogels with a high water content, excellent protein compatibility and high protein loading, have emerged as attractive vehicles for protein delivery.13-16 For example, van Nostrum et al. immobilized the model antigen ovalbumin in dextran-based cationic nanogels, which enhanced uptake of the antigen by dendritic cells.17 Wu et al. prepared crosslinked physical/chemical composite nanogels for co-delivery of doxorubicin, interleukin-2 and interferon γ for synergistic anticancer effects.18 We investigated photo-click hyaluronic acid (HA) nanogels for loading of CC and granzyme B and targeted protein therapy of human ovarian cancer, breast cancer and

2 ACS Paragon Plus Environment

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orthotopic lung cancer.15,19,20 It should be noted, however, that chemical nanogels typically require use of catalyst, initiator, enzyme or photo irradiation that potentially leads to toxicity concerns or protein denaturation.21 Another issue with chemical nanogels is complex synthesis and possible cross-reaction between nanogel precursors and protein drugs.22 On the contrary, physical nanogels e.g. crosslinked via ionic, hydrophobic or hydrogen bonding interactions usually possess better biocompatibility and less protein damage.23 However, few reported physical nanogels display good stability, selective and efficient cell uptake, fast endosomal escape, and triggered intracellular protein release that are key to intracellular protein delivery. α-Helical coiled coils are unique protein structural motifs that are composed of two or more α-helical peptides running in parallel or antiparallel direction and wrapping around each other in a heptad repeat fashion of (abcdefg)n. In this heptad repeat, positions a and d typically contain hydrophobic residues forming tight “knobs-in-holes” packing, while positions e and g are mainly occupied by complementary charged amino acids to form inter-chain salt bridges.24,25 Investigations indicated the wide applications of coiled-coil motifs in advanced drug

delivery

systems,26,27

including

host-guest

hybrid

polymer

therapeutics,28

polymer-peptide conjugates,29 drug-free macromolecular therapeutics30,31 and self-assembling cages.32 In particular, pH-sensitive E3/K3 coiled-coil motifs have drawn remarkable attention for the construction of nanomedicines owing to their high structural stability at extracellular neutral pH condition and pH-triggered unfolding behaviors at late endosomal environment (pH 5.0).33-35 For example, Mastrobattista et al. employed E3/K3 coiled coils to modify liposomes for targeted delivery of nucleic acid, resulting in significantly improved transfection efficiency.36 Klok et al. conjugated either E3 or K3 to poly(N-(2-hydroxypropyl) methacrylamide) to prepare complementary polymer-drug conjugates for pH-triggered doxorubicin release.37 In addition, E3/K3 coiled coils have also been used for selective labeling of live cell surface proteins for real-time cell fluorescence imaging.38-40



In this paper, we report pH-sensitive E3/K3 coiled-coil peptide-crosslinked hyaluronic acid nanogels (HA-cNGs) for targeted intracellular protein delivery to CD44 overexpressing MCF-7 breast cancer cells (Scheme 1). Surprisingly, HA-cNGs loaded with saporin, a cell impermeable ribosome inactivating protein originated from the seeds of Saponaria officinalis,41 showed a high antitumor efficacy against MCF-7 human breast cancer cells. These coiled-coil peptide-crosslinked hyaluronic acid nanogels have a great potential for intracellular protein delivery.

Scheme 1. Illustration of the preparation of pH-sensitive coiled-coil peptide-crosslinked hyaluronic acid nanogels (HA-cNGs) for efficient intracellular delivery of saporin. 4 ACS Paragon Plus Environment

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2. EXPERIMENTAL METHODS 2.1 Synthesis of HA-E3 and HA-K3 Conjugates Coiled-coil peptide (E3 or K3) was conjugated to HA (35 kDa) using 2-maleimido ethylamine hydrochloride (MEA·HCl) as a linker. Taking the synthesis of HA-K3 as an example, HA (500 mg, 14 nmol), MEA·HCl (139 mg, 790 nmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)4-methylmorpholinium chloride (DMTMM, 219 mg, 790 nmol) were dissolved in 30 mL of 2-(N-morpholino) ethanesulfonic acid (MES) solution (20 mM, pH 6.8). The mixture was stirred at room temperature (RT) for 10 h. The resulting product HA-MEA was purified by extensive dialysis (Spectra/Pore, MWCO 3500) against de-ionized (DI) water, followed by lyophilization. 1H NMR (D2O) analysis showed a MEA/HA molar ratio of 12 (Figure 1). Next, HA-MEA (100 mg, 2.7 nmol) and K3 (58.09 mg, 21.6 nmol, K3/HA-MEA = 8/1, mol/mol) separately dissolved in phosphate buffer (PB, 100 mM, pH 7.0) were mixed and reacted under stirring at 40 oC overnight. The unreacted MEA on HA was terminated with excessive cysteine. HA-K3 was purified by extensive dialysis (Spectra/Pore, MWCO 14000) and lyophilization. Yield: 146.8 mg (92.9 %). The amount of K3 conjugated on HA was quantified by bicinchoninic acid (BCA) assay (n=3). HA-E3 was synthesized in the same manner.

2.2 Preparation of pH-Sensitive Coiled-Coil Peptide-Crosslinked Hyaluronic Acid Nanogels pH-Sensitive coiled-coil peptide-crosslinked hyaluronic acid nanogels (HA-cNGs) were prepared via nanoprecipitation. Briefly, equivalent HA-E3 and HA-K3 solutions (0.2 mg/mL) in PB (5 mM, pH 7.0) were rapidly mixed using a double-chamber mixing syringe and added to 10 mL of acetone (1/20, v/v) immediately via a microflow syringe pump (0.125 mL/min, KDS-101, Kd Scientific, USA) at ambient temperature without stirring, followed by 10 min



static incubation. The acetone was removed by vacuum rotary evaporation, and the resulting nanogel suspension was purified by extensive dialysis (Spectra/Pore MWCO 3500) against PB (10 mM, pH 7.4) for 12 h. The size distribution and morphology of nanogels were measured by DLS and TEM, respectively. Furthermore, the formation of coiled-coil motif between E3 and K3 was studied by CD analysis.

2.3 Colloidal Stability and pH-Sensitivity of HA-cNGs To verify the colloidal stability of HA-cNGs under physiological conditions, CD spectra of HA-cNGs in DI water, 10 mM PB (pH 7.4) and 10 mM PB containing 150 mM NaCl (pH 7.4) were measured after 4 h incubation. CD measurements were also used to investigate the cell-free pH-sensitivity of HA-cNGs, which was carried out in 10 mM of PB at simulated pH conditions of extracellular (pH 7.4), early endosome (pH 6.0), and late endosome (pH 5.0) incubated at 37 oC in a shaking bed for 1 h or 10 h. The pH-triggered disassembly of coiled-coil motif of HA-cNGs in acidic condition was further confirmed by determining the size changes of HA-cNGs using DLS.

2.4 In Vitro Antitumor Efficacy of HA-cNGs-SAP The in vitro antitumor efficacy of HA-cNGs-SAP against CD44 overexpressing MCF-7 human breast cancer cells was evaluated by MTT assay. In brief, the cells were plated in a 96-well plate (80 µL, 5000 cells/well) using DMEM medium supplemented with 10% fetal bovine serum in an atmosphere containing 5% CO2 at 37 °C for 24 h. 20 µL of HA-cNGs-SAP samples with prescribed concentrations were added. The cells were cultured for 4 h. The supernant was removed and replaced by an equal volume of fresh DMEM medium. The cells were further cultured for 68 h. 10 µL of MTT stock solution (5 mg/mL, in PBS) was added to each well. The cells were kept in dark and further incubated with MTT for 4 h. The supernant


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was carefully discarded, and 150 µL of DMSO was added to each well to dissolve the purple formazan crystals by placing the 96-well plate in a shaking bed for 15 min. The absorbance of each well was then measured using a microplate reader (Bio-Tek, ELX 808IU) at a wavelength of 492 nm, and the wells containing DMSO only were used as the background noise. The relative cell viability (%) was calculated by comparing the absorbance of each sample at 492 nm with PBS treated cells (without exposure to HA-cNGs-SAP). The data were presented as mean ± SD (n =4). The apoptosis induction of MCF-7 cells by HA-cNGs was evaluated using flow cytometry by staining MCF-7 cells with an Annexin V-FITC/propidium iodide (PI) kit (MesGEN, China). MCF-7 cells were digested by EDTA-free trypsin, collected with centrifugation, and washed by PBS. The cells were stained by 100 µL of mixing buffer of Annexin V-FITC and binding buffer (0.8/100, v/v) for 15 min at ambient temperature in dark, and further stained by 100 µL of mixing buffer of PI and binding buffer (1/100, v/v) for 5 min at 4 °C. The double-stained cells were centrifuged and washed by PBS, re-suspended in 500 µL of binding buffer, and measured immediately by flow cytometry (Becton Dickinson, FACS Canto, USA).

3. RESULTS AND DISCUSSION 3.1 Synthesis of HA-E3 and HA-K3 Conjugates and Coiled-Coil Nanogels HA-E3 and HA-K3 conjugates were readily synthesized from 35 kDa HA using MEA·HCl as a linker (Scheme 2). 1H NMR spectrum of HA-MEA showed signals at δ 1.86−2.01, 3.11−4.19, and 4.38-4.64 attributable to HA backbone as well as resonance at δ 6.88 to the maleimide protons of MEA moiety (Figure 1). The comparison of the integrals of signals at δ 6.88 and 4.50 (anomeric protons on HA backbone) revealed an average of 12 MEA molecules per HA chain, corresponding to a degree of substitution (DS) of 13. E3 and K3 peptides were



conjugated to HA-MEA at a theoretical molar ratio of 8 via thiol-maleimide addition. The unreacted maleimide groups were terminated with cysteine. BCA assays confirmed that HA-E3 and HA-K3 had actual peptide/HA molar ratios of 7.5 and 7.8, which corresponded to a conjugation efficiency of 93.8% and 97.5%, respectively. As a result, HA-E3 conjugate has a peptide content of 36.3 wt.% and HA-K3 has a peptide content of 37.2 wt.%. We found that large aggregates formed during the conjugation process when K3/HA molar ratio was more than 8, probably caused by the strong interactions between negatively charged HA and positively charged K3 peptide. Even at a high salt condition (up to 150 mM), aggregation took place when K3/HA molar ratio was over 8.

Scheme 2. Synthesis of HA-E3 and HA-K3 conjugates (with HA-E3 as an example).


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H2O ~

b

HA, d, e

a

c

7

6

5

4

3

2

1

0

Chemical Shift (ppm) Figure 1. 1H NMR spectrum (400 MHz, D2O) of HA-MEA.

Nanogels were conveniently prepared through a nanoprecipitation method.42 To this end, solutions of HA-E3 and HA-K3 (1/1, mol/mol) in PB (HA-peptide concentration: 0.02 wt.%) were injected into acetone (1/20, v/v) through a double-chamber mixing syringe. DLS measurements exhibited that the resulting nanogels (HA-cNGs) had a hydrodynamic diameter of about 176 nm (Figure 2). TEM analysis revealed that HA-cNGs possessed a spherical


Biomacromolecules

morphology and a size comparable to that determined by DLS.

16

12

Intensity (%)

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

8 200 nm

4

0 100

1000 Size (nm)

Figure 2. Size distribution of HA-cNGs measured by DLS measurement. Inset: TEM image of HA-cNGs.

Circular dichroism (CD) measurements indicated that the E3 and K3 peptides in the individual HA-E3 and HA-K3 conjugates had a random coil structure in aqueous media (Figure 3A), in accordance with previous reports.37 In contrast, CD analysis of the HA-cNGs revealed minima at 208 and 222 nm with similar intensities (Figure 3A), which are characteristic for the formation of α-helical coiled-coil motifs.43 The large molar ellipticity indicated a high content of coiled-coil motifs formed by E3 and K3 association. The colloidal stability of HA-cNGs in solutions with different ionic strengths was also evaluated by CD measurements. As shown in Figure 3B, the characteristic coiled coil CD signature remained unchanged under 10 mM PB containing 150 mM NaCl condition, indicating that HA-cNGs have good stability under physiological conditions.44 Interestingly, the molar ellipticity of HA-cNGs was reduced with decreasing pH from 7.4 to 6.0 (Figure 3C), supporting that HA-cNGs are pH-sensitive and α-helical coiled-coil content decreases at acidic pH.35


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Decreasing the pH to 5 results in a significant decrease in the intensity of the coiled coil characteristic CD signals, indicative of unfolding and dissociation of part of the E3/K3 superstructures. In accordance, DLS measurements showed that HA-cNGs swelled from 176 nm to ca. 345 and 528 nm following 10 h incubation at pH 6.0 and 5.0, respectively (Figure 3D). These results confirm that our HA-cNGs have good extracellular stability and are prone to disassembly in acidic condition, which renders them promising for pH-triggered intracellular delivery of therapeutic proteins. 40

0 -20

210 225 240 Wavelength (nm)

40

[Θ]×10-3(deg⋅⋅cm2⋅dmol-1)

20

-40 195

C

B

HA-E3 HA-K3 HA-cNGs

D

40

DI H2O 10 mM PB 10 mM PB, 150 mM NaCl

20 0 -20 -40 195

255


24

pH 6.0

20

pH 7.4

0

18 12 6

-20 -40

255

pH 7.4, 1 h pH 7.4, 10 h pH 6.0, 1 h pH 6.0, 10 h pH 5.0, 1 h pH 5.0, 10 h

pH 5.0

Intensity (%)

[Θ]×10-3 (deg⋅⋅cm2⋅dmol-1)

A

[Θ]×10-3 (deg⋅⋅cm2⋅dmol-1)

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

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195


255

0 100

1000 Size (nm)

10000

Figure 3. (A) CD spectra of HA-E3, HA-K3 and HA-cNGs (equimolar mixture of HA-E3 and HA-K3) measured in 10 mM PB at pH 7.4; (B) CD characterization of HA-cNGs under different conditions; (C) pH-responsivity of HA-cNGs in 10 mM PB studied by CD. HA-cNGs were incubated for 10 h at pH 7.4, 6.0 or 5.0 at 37 oC; (D) pH-triggered size change of HA-cNGs in 10 mM PB over time at 37 oC. All samples have the same peptide concentration (50 µM).



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3.2 Loading and pH-Triggered Release of Cytochrome C Cy5-labeled cytochrome C (Cy5-CC) was used as a model protein to investigate the protein loading and release behaviors of HA-cNGs. The results, which are summarized in Table 1, showed good loading of Cy5-CC, in which protein loading content (PLC) of 1.0 to 4.0 wt.% was obtained at theoretical PLC of 1 to 5 wt.%, respectively. The protein loading efficiency (PLE) was over 99% at a theoretical PLC of 1 wt.%, 94.9% at 2 wt.% and 78.7% at 5 wt.%, respectively. Interestingly, the size of Cy5-CC loaded HA-cNGs decreased from 175 nm to 165 nm with increasing PLC from 1 wt.% to 4 wt.%, which could be caused by strong interactions, including ionic interaction and hydrogen bonding, between positively charged Cy5-CC and negatively charged HA backbone. Cy5-CC loaded HA-cNGs had decent polydispersity index (PDI) of 0.12~0.18. Zeta potential measurements displayed that Cy5-CC loaded HA-cNGs had negative surface charges from -29.7 to -26.5 mV, similar to those reported for HA nanogels and HA-coated nanoparticles.45,46

Table 1. Characterization of Cy5-CC loaded HA-cNGs in PB (pH 7.4, 10 mM). PLC (wt. %) Entry

a

PLE (%)

Sizeb (nm)

PDIb

Zetac (mV)

Theory

Determined

1

0

-

-

176 ± 2

0.22

-30.3 ± 0.5

2

1

1.0

>99

175 ± 1

0.15

-29.7 ± 0.4

3

2

1.9

94.9

168 ± 3

0.12

-28.2 ± 0.3

4

5

4.0

78.7

165 ± 2

0.18

-26.5 ± 0.6

a

Determined by fluorescence spectrometry at 25 oC in PB.

b

Determined by Zetasizer Nano-ZS at 25 oC in PB.

c

Determined by Zetasizer Nano-ZS equipped with a standard capillary electrophoresis cell at

25 oC in DI water.


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The pH triggered protein release from HA-cNGs-CC was studied in 10 mM PB at 37 oC at varying pH of 7.4, 6.0 and 5.0, respectively. Figure 4 shows that only 18.4% of protein was released after 24 h at pH 7.4, confirming that HA-cNGs-CC has decent stability and low premature protein release at physiological pH condition. Interestingly, 76.8% of Cy5-CC was released in 24 h at pH 6.0, likely as a result of partial unfolding of coiled-coil motifs. At pH 5.0, protein release rate was further increased with more than 85% of protein release in 12 h and over 91% in 24 h. Notably, E3/K3 peptides coiled-coil nanogels show a faster pH-triggered

protein

release

behavior

than

previously

reported

pH-responsive

nano-systems.47-49 Moreover, unlike pH-sensitive chemical nanogels based on e.g. acid-cleavable hydrazone and acetal linkers, these coiled-coil nanogels does not generate any by-products that might cause toxicity concerns. These results show that HA-cNGs are highly pH-sensitive and fast protein release can be achieved at endo/lysosomal pH.

100 Cummulative Release (%)

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

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pH 7.4 pH 6.0 pH 5.0

80 60 40 20 0

0

5

10 15 Time (h)

20

25

Figure 4. pH-triggered protein release profiles of HA-cNGs-CC in 10 mM PB at pH 7.4, 6.0 and 5.0, respectively.

3.3 CD44 Mediated Cellular Uptake and Intracellular Protein Release The antitumor potential of protein loaded HA-cNGs is closely dependent on their cellular



uptake and endosomal escape capability. The intracellular delivery of Cy5-CC loaded HA-cNGs was monitored in CD44 overexpressing MCF-7 human breast cancer cells using confocal laser scanning microscopy (CLSM). Obvious red fluorescence of Cy5 was observed in MCF-7 cells after 4 h incubation with HA-cNGs-CC at 37 oC (Figure 5A), indicating efficient cellular uptake of HA-cNGs. Much stronger intracellular Cy5 fluorescence was detected when increasing incubation time to 8 h. The cellular uptake of Cy5-CC loaded HA-cNGs was, however, greatly inhibited by pretreating MCF-7 cells with free HA (5 mg/mL), supporting that HA-cNGs are mainly internalized by MCF-7 cells via receptor-mediated pathway.20 In comparison, little Cy5 fluorescence was detected in MCF-7 cells following 8 h incubation with free Cy5-CC. The cellular uptake behaviors of HA-cNGs-CC by MCF-7 cells were quantitatively evaluated by flow cytometry. Figure 5B shows efficient uptake of HA-cNGs-CC and competitive inhibition by free HA, in which 3.7and 4.2-fold reduction of cellular uptake was observed following additional 2 and 4 h culture, respectively.


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A

DAPI DAPI

HA

-

HA

H

H A+

A

scNG

G -c N

G -c N

e Fre

Cy5

Merge Merge

CC 4h

C s -C 8h

s -C

C 8h

-C Cy5

C

8h

B

PBS

120

2h 4h HA + 2 h HA + 4 h

80

Counts

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

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40

0 1

10

100 1000 Cy5 Fluorescence Intensity

10000

Figure 5. Targeted cellular uptake of HA-cNGs-CC by CD44 overexpressing MCF-7 human breast cancer cells monitored by CLSM (A) and flow cytometry (B). Competitive inhibition



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experiment was carried out by pretreating MCF-7 cells with free HA (5 mg/mL) for 4 h prior to HA-cNGs incubation. (Bar: 20 µm)

For therapeutic proteins that act in the cytoplasm, endosomal escape capability is of great importance. Previous report showed that coiled-coil motifs can drive artificial or bio-membrane fusion,50 which would likely result in enhanced endosomal escape of coiled coil-based nanosystems. CLSM observations showed that Cy5-CC mostly co-localized with late endosomes/lysosomes of MCF-7 cells at 4 h incubation (Figure 6). At 8 h, red fluorescence owing to Cy5-CC was observed in the merged image, indicating that part of Cy5-CC has escaped from endosomes.51 Much stronger red fluorescence was discerned at 12 h. These results indicate that HA-cNGs can effectively escape from endo/lysosomes. The high endosome disruption activity of HA-cNGs is likely due to the fact that disassembly of HA-cNGs in acidic endosomes leads to free HA-E3 and HA-K3 chains that promote fusion with endosomal membranes.

DAPI

Cy5

Lysotracker

Merge

I 4h

II 8h

III 12 h

Figure 6. Endosomal escape of HA-cNGs-CC in MCF-7 cells following 4 h, 8 h or 12 h


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incubation observed by confocal microscopy. (Bar: 20 µm)

3.4 In Vitro Antitumor Activity of Saporin-Loaded HA-cNGs To evaluate their antitumor potential, we loaded saporin (SAP), a highly potent protein toxin,52 as a therapeutic model protein to HA-cNGs. Interestingly, MTT assays showed that SAP-loaded HA-cNGs induced a high antitumor effect to MCF-7 cells with a remarkably low half-maximal inhibitory concentration (IC50) of 12.2 nM (Figure 7A), which is comparable to that reported for saporin-loaded lipid-like nanoparticles against B16F10 murine melanoma cells and saporin-based immunotoxin for human leukaemia and lymphoma cells.53,54 In contrast, free SAP at ~ 12.2 nM demonstrated little cell growth inhibition to MCF-7 cells, likely due to poor cellular uptake.53,55 Notably, the in vitro antitumor efficacy of HA-cNGs-SAP was greatly inhibited by pretreating MCF-7 cells with free HA (5 mg/mL), further supporting that SAP-loaded HA-cNGs are taken up by MCF-7 cells in a receptor-mediated mechanism. We further investigated the in vitro antitumor activity of HA-cNGs-SAP using Annexin V-FITC/PI staining assays.56 Flow cytometry analyses indicated that free SAP induced negligible cell apoptosis while HA-cNGs-SAP caused 19.2% cell apoptosis (Figure 7B). The pretreatment of MCF-7 cells with free HA led to 2.9-fold reduction of cell apoptosis, further supporting the active targeting effect of HA-cNGs-SAP to MCF-7 cells. Figure 7C shows that blank HA-cNGs were essentially non-cytotoxic toward MCF-7 cells even at a concentration of 1 mg/mL. Hence, coiled-coil peptide-crosslinked hyaluronic acid nanogels have appeared to be a novel, nontoxic and versatile platform for intracellular protein delivery.


Biomacromolecules

C

A

100

Cell Viability (%)

Cell Viability (%)

100 75 50 25 0

Free Saporin HA-cNGs HA-cNGs with Free HA

80 60 40 20

1

10

00

100

Saporin Concentration (nM)

0.01 0.2 0.1 0.5 1.0 0.01 0.1 0.2 0.5 1.0 HA-cNGs Concentration (mg/mL)

B HA-cNGs-SAP 104

9.0%

PI

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

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PBS

SAP

HA+HA-cNGs-SAP 104

104

104

1.6%

103

103

103

103

102

102

102

102 101

101

10.2%

101

5.1%

101

100 100 101 102

103 104

100 100 101 102

103 104

100 100 101 102

103 104

100 100 101 102

103 104

Annexin V-FITC

Figure 7. Cell growth inhibition of HA-cNGs-SAP in CD44 overexpressing MCF-7 human breast cancer cells evaluated by MTT assays (A) and Annexin V-FITC/PI staining assays (B); (C) Cytotoxicity of blank HA-cNGs against MCF-7 cells.

4. CONCLUSIONS We have demonstrated that pH-sensitive E3/K3 coiled-coil peptide crosslinked hyaluronic acid nanogels (HA-cNGs) mediate efficient intracellular protein delivery to CD44 overexpressing MCF-7 human breast cancer cells. Interestingly, HA-cNGs show decent protein loading, good colloidal stability under physiological conditions and fast response to mildly acidic pH. Flow cytometry and confocal studies reveal that protein-loaded HA-cNGs are efficiently taken up by MCF-7 cells through a receptor mediated pathway. Moreover, HA-cNGs can help proteins to escape from endosomes, likely via fusion of uncoiled E3/K3 peptides with endosomal membrane. Saporin-loaded HA-cNGs are highly potent against


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MCF-7 cells with a remarkably low IC50 of 12.2 nM. These pH-sensitive coiled-coil peptide crosslinked hyaluronic acid nanogels with easy preparation, active tumor-targeting ability, and efficient intracellular protein release have appeared as a smart approach for cancer protein therapy.

ASSOCIATED CONTENT Supporting

Information.

Detailed

experimental

procedures

including

materials,

characterization, and loading and pH-triggered release of protein. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Authors *Tel:

+86-512-65880098.

E-mail:

[email protected]

(J.

Zhang);

[email protected] (Z. Zhong)

ACKNOWLEDGMENTS This work was supported by research grants from the National Natural Science Foundation of China (NSFC 51403147, 51633005).

REFERENCES (1) Lu, Y.; Sun, W.; Gu, Z. J. Control. Release 2014, 194, 1-19. (2) Mitragotri, S.; Burke, P. A.; Langer, R. Nat. Rev. Drug Discov. 2014, 13 (9), 655-672. (3) Yu, M.; Wu, J.; Shi, J.; Farokhzad, O. C. J. Control. Release 2016, 240, 24-37. (4) Zhao, M.; Hu, B.; Gu, Z.; Joo, K.-I.; Wang, P.; Tang, Y. Nano Today 2013, 8 (1), 11-20. (5) Zhao, M.; Liu, Y.; Hsieh, R. S.; Wang, N.; Tai, W.; Joo, K. I.; Wang, P.; Gu, Z.; Tang, Y. J. Am. Chem. Soc. 2014, 136 (43), 15319-15325. (6) Prasetyanto, E. A.; Bertucci, A.; Septiadi, D.; Corradini, R.; Castro-Hartmann, P.; De Cola, L. Angew. Chem. Int. Ed. 2016, 55 (10), 3323-3327.



(7) Jiang, Y.; Lu, H.; Chen, F.; Callari, M.; Pourgholami, M.; Morris, D. L.; Stenzel, M. H. Biomacromolecules 2016, 17 (3), 808-817. (8) Lee, Y.; Ishii, T.; Cabral, H.; Kim, H. J.; Seo, J. H.; Nishiyama, N.; Oshima, H.; Osada, K.; Kataoka, K. Angew. Chem. Int. Ed. 2009, 48 (29), 5309-5312. (9) Yang, W.; Xia, Y.; Zou, Y.; Meng, F.; Zhang, J.; Zhong, Z. Chem. Mater. 2017, 29 (20), 8757-8765. (10) Molina, M.; Asadian-Birjand, M.; Balach, J.; Bergueiro, J.; Miceli, E.; Calderón, M. Chem. Soc. Rev. 2015, 44 (17), 6161-6186. (11) Vermonden, T.; Censi, R.; Hennink, W. E. Chem. Rev. 2012, 112 (5), 2853-2888. (12) Tahara, Y.; Mukai, S. A.; Sawada, S.; Sasaki, Y.; Akiyoshi, K. Adv. Mater. 2015, 27 (34), 5080-5088. (13) Kawasaki, R.; Sasaki, Y.; Katagiri, K.; Mukai, S. a.; Sawada, S. i.; Akiyoshi, K. Angew. Chem. Int. Ed. 2016, 55 (38), 11377-11381. (14) Li, D.; van Nostrum, C. F.; Mastrobattista, E.; Vermonden, T.; Hennink, W. E. J. Control. Release 2017, 259, 16-28. (15) Chen, J.; Ouyang, J.; Chen, Q.; Deng, C.; Meng, F.; Zhang, J.; Cheng, R.; Lan, Q.; Zhong, Z. ACS Appl. Mater. Interfaces 2017, 9 (28), 24140-24147. (16) Liang, K.; Ng, S.; Lee, F.; Lim, J.; Chung, J. E.; Lee, S. S.; Kurisawa, M. Acta Biomater. 2016, 33 (1), 142-152. (17) Li, D.; Kordalivand, N.; Fransen, M. F.; Ossendorp, F.; Raemdonck, K.; Vermonden, T.; Hennink, W. E.; Van Nostrum, C. F. Adv. Funct. Mater. 2015, 25 (20), 2993-3003. (18) Wu, X.; He, C.; Wu, Y.; Chen, X.; Cheng, J. Adv. Funct. Mater. 2015, 25 (43), 6744-6755. (19) Chen, J.; Zou, Y.; Deng, C.; Meng, F.; Zhang, J.; Zhong, Z. Chem. Mater. 2016, 28 (23), 8792-8799. (20) Li, S.; Zhang, J.; Deng, C.; Meng, F.; Yu, L.; Zhong, Z. ACS Appl. Mater. Interfaces 2016, 8 (33), 21155-21162. (21) Wu, W.; Yao, W.; Wang, X.; Xie, C.; Zhang, J.; Jiang, X. Biomaterials 2015, 39, 260-268. (22) Patenaude, M.; Campbell, S.; Kinio, D.; Hoare, T. Biomacromolecules 2014, 15 (3), 781-790. (23) Raemdonck, K.; Demeester, J.; De Smedt, S. Soft Matter 2009, 5 (4), 707-715. (24) Lupas, A. N.; Bassler, J. Trends Biochem. Sci. 2017, 42 (2), 130-141. (25) Pechar, M.; Pola, R.; Laga, R.; Braunova, A.; Filippov, S. K.; Bogomolova, A.; Bednarova, L.; Vanek, O.; Ulbrich, K. Biomacromolecules 2014, 15 (7), 2590-2599. (26) Wu, Y.; Collier, J. H. WIREs Nanomed. Nanobiotechnol. 2017, 9 (2). (27) Apostolovic, B.; Danial, M.; Klok, H. A. Chem. Soc. Rev. 2010, 39 (9), 3541-3575. (28) Malashkevich, V. N.; Higgins, C. D.; Almo, S. C.; Lai, J. R. Biopolymers 2015, 104 (3), 178-185. (29) Shu, J. Y.; Panganiban, B.; Xu, T. Annu. Rev. Phys. Chem. 2013, 64 (1), 631-657. (30) Kverka, M.; Hartley, J. M.; Chu, T.-W.; Yang, J.; Heidchen, R.; Kopeček, J. Biomaterials 2014, 20 ACS Paragon Plus Environment

Page 20 of 23

Page 21 of 23 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

35 (22), 5886-5896. (31) Zhang, R.; Yang, J.; Chu, T. W.; Hartley, J. M.; Kopeček, J. Adv. Healthc. Mater. 2015, 4 (7), 1054-1065. (32) Fletcher, J. M.; Harniman, R. L.; Barnes, F. R. H.; Boyle, A. L.; Collins, A.; Mantell, J.; Sharp, T. H.; Antognozzi, M.; Booth, P. J.; Linden, N.; Miles, M. J.; Sessions, R. B.; Verkade, P.; Woolfson, D. N. Science 2013, 340 (6132), 595-599. (33) Thomas, F.; Burgess, N. C.; Thomson, A. R.; Woolfson, D. N. Angew. Chem. Int. Ed. 2016, 55 (3), 987-991. (34) Gormley, A. J.; Chandrawati, R.; Christofferson, A. J.; Loynachan, C.; Jumeaux, C.; Artzy-Schnirman, A.; Aili, D.; Yarovsky, I.; Stevens, M. M. Chem. Mater. 2015, 27 (16), 5820-5824. (35) Reja, R. M.; Khan, M.; Singh, S. K.; Misra, R.; Shiras, A.; Gopi, H. N. Nanoscale 2016, 8 (9), 5139-5145. (36) Oude Blenke, E. E.; van den Dikkenberg, J.; van Kolck, B.; Kros, A.; Mastrobattista, E. Nanoscale 2016, 8 (16), 8955-8965. (37) Apostolovic, B.; Deacon, S. P.; Duncan, R.; Klok, H.-A. Biomacromolecules 2010, 11 (5), 1187-1195. (38) Yano, Y.; Furukawa, N.; Ono, S.; Takeda, Y.; Matsuzaki, K. Biopolymers 2016, 106 (4), 484-490. (39) Reinhardt, U.; Lotze, J.; Zernia, S.; Morl, K.; Beck-Sickinger, A. G.; Seitz, O. Angew. Chem. Int. Ed. 2014, 53 (38), 10237-10241. (40) Reinhardt, U.; Lotze, J.; Morl, K.; Beck-Sickinger, A. G.; Seitz, O. Bioconjugate Chem. 2015, 26 (10), 2106-2117. (41) Giansanti, F.; Sabatini, D.; Pennacchio, M. R.; Scotti, S.; Angelucci, F.; Dhez, A. C.; Antonosante, A.; Cimini, A.; Giordano, A.; Ippoliti, R. J. Cell. Biochem. 2015, 116 (7), 1256-1266. (42) Steinhilber, D.; Witting, M.; Zhang, X.; Staegemann, M.; Paulus, F.; Friess, W.; Kuchler, S.; Haag, R. J. Control. Release 2013, 169 (3), 289-295. (43) Deacon, S. P. E.; Apostolovic, B.; Carbajo, R. J.; Schott, A.-K.; Beck, K.; Klok, H.-A.; Duncan, R. Biomacromolecules 2011, 12 (1), 19-27. (44) Apostolovic, B.; Klok, H.-A. Biomacromolecules 2008, 9 (11), 3173-3180. (45) Zhao, Q.; Geng, H.; Wang, Y.; Gao, Y.; Huang, J.; Wang, Y.; Zhang, J.; Wang, S. ACS Appl. Mater. Interfaces 2014, 6 (22), 20290-20299. (46) Zhong, Y.; Goltsche, K.; Cheng, L.; Xie, F.; Meng, F.; Deng, C.; Zhong, Z.; Haag, R. Biomaterials 2016, 84, 250-261. (47) Calderon, M.; Welker, P.; Licha, K.; Fichtner, I.; Graeser, R.; Haag, R.; Kratz, F. J. Control. Release 2011, 151 (3), 295-301. (48) Hammer, N.; Brandl, F. P.; Kirchhof, S.; Goepferich, A. M. J. Control. Release 2014, 183, 67-76. (49) Lin, Z.; Li, J.; He, H.; Kuang, H.; Chen, X.; Xie, Z.; Jing, X.; Huang, Y. RSC Adv. 2015, 5 (13), 21 ACS Paragon Plus Environment


9546-9555. (50) Zheng, T.; Voskuhl, J.; Versluis, F.; Zope, H. R.; Tomatsu, I.; Marsden, H. R.; Kros, A. Chem. Commun. 2013, 49 (35), 3649-3651. (51) Kim, H. J.; Ishii, A.; Miyata, K.; Lee, Y.; Wu, S.; Oba, M.; Nishiyama, N.; Kataoka, K. J. Control. Release 2010, 145 (2), 141-148. (52) Lai, P. S.; Pai, C. L.; Peng, C. L.; Shieh, M. J.; Berg, K.; Lou, P. J. J. Biomed. Mater. Res. A 2008, 87 (1), 147-155. (53) Wang, M.; Alberti, K.; Sun, S.; Arellano, C. L.; Xu, Q. Angew. Chem. Int. Ed. 2014, 126 (11), 2937-2942. (54) Holmes, S. E.; Bachran, C.; Fuchs, H.; Weng, A.; Melzig, M. F.; Flavell, S. U.; Flavell, D. J. Immunopharm. Immunot. 2015, 37 (1), 42-55. (55) Bostad, M.; Olsen, C. E.; Peng, Q.; Berg, K.; Hogset, A.; Selbo, P. K. J. Control. Release 2015, 206, 37-48. (56) Law, B. Y. K.; Chan, W. K.; Xu, S. W.; Wang, J. R.; Bai, L. P.; Liu, L.; Wong, V. K. W. Sci. Rep. 2014, 4, 5510-5534.


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Table of Contents Graphic

pH-Sensitive Coiled-Coil Peptide-Crosslinked Hyaluronic Acid Nanogels: Synthesis and Targeted Intracellular Protein Delivery to CD44 Positive Cancer Cells

Lingling Ding, Yu Jiang, Jian Zhang,* Harm-Anton Klok, Zhiyuan Zhong *


pH-Sensitive Coiled-Coil Peptide-Cross-Linked Hyaluronic Acid

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