A Smart H2O2-Responsive Drug Delivery System Made by Halloysite

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A Smart HO-Responsive Drug Delivery System Made by Halloysite Nanotubes and Carbohydrate Polymers Feng Liu, Libin Bai, Hailei Zhang, Hongzan Song, Liandong Hu, Yonggang Wu, and Xinwu Ba ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10867 • Publication Date (Web): 01 Sep 2017 Downloaded from http://pubs.acs.org on September 1, 2017

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ACS Applied Materials & Interfaces

A Smart H2O2-Responsive Drug Delivery System Made by Halloysite Nanotubes and Carbohydrate Polymers Feng Liu, a Libin Bai,a Hailei Zhang,a* Hongzan Songa, Liandong Hu,b Yonggang Wu,a* and Xinwu Baa

a

College of Chemistry & Environmental Science, Hebei University, Baoding, P. R.

China. b

School of Pharmaceutical Science, Hebei University, Baoding, P. R. China.

Corresponding authors: No.180 Wusi Road, College of Chemistry & Environmental Science, Hebei University, Baoding, Hebei Province, 071002, P. R. China. E-mails: [email protected] (Hailei Zhang) [email protected] (Yonggang Wu)

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Abstract: A

novel

chemical

hydrogel

was

facilely

achieved

by

coupling

1,4-phenylenebisdiboronic acid-modified halloysite nanotubes (HNTs-BO) with compressible starch. The modified HNTs and prepared hydrogel were characterized by solid-state nuclear magnetic resonance (NMR), Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and transmission electron microscope (TEM). The linkage of B-C in the hydrogel can be degraded into B-OH and C-OH units in the presence of H2O2, and result into the degradation of the chemical hydrogel. Pentoxifylline was loaded into the lumen of HNTs-BO and then give the pentoxifylline-loaded hydrogel. Drug release profile shows that it was no more than 7% dissolved when using PBS solution as release medium. Notably, a completely released (near 90%) can be achieved with the addition of H2O2 ([H2O2] =1×10-4 M), suggesting a high H2O2-responsiveness of as-formed hydrogel. The drug release results also show that the “initial burst release” can be effectively suppressed by loading pentoxifylline inside the lumen of HNTs than embedding the drug in hydrogel network. The drug-loaded hydrogel with H2O2-responsive release behaviour may open up a broader application in the field of biomedicine. Key words: halloysite nanotubes, hydrogel, H2O2-responsiveness, drug delivery, initial burst release

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1. Introduction Halloysite nanotube (HNT) is natural aluminosilicate clay mineral, which has the same chemical composition as kaolinite (Al2Si2O5(OH)4 nH2O) 1-2. The multilayers of hollow nanotubes are formed by curved and rolled up of the adjacent alumina and silica sheets along with their water of hydration 3-4. The Al−OH groups are distributed on internal surface of HNTs, whereas the Si-O-Si groups are overspread on the external surface

5-6

. Benefiting from the hollow tubular shape and the large cavity

volume, HNTs can be used as the desirable natural nano-carriers for loaded active agents

7-12

. Additionally, several recent reports demonstrate the safety and

biocompatibility of HNTs. The cytotoxicity tests conducted by Vergaro et al indicated that the viabilities of human breast cancer cells and human epithelial adenocarcinoma cells were preserved after treating with HNTs (up to 0.075 mg mL-1) 13. Fakhrullina et al demonstrated that HNTs does not induce severe toxic effects on the organisms of Caenorhabditis elegans

14

. Kryuchkova et al reported that HNTs exhibited very low

toxicity towards Paramecium caudatum

15

. The attractive properties including

biocompatibility and nontoxicity, as well as non-degradation, hydrophilic and low-cost also make HNTs be promising materials in the field of biochemistry and biomedicine 16-21. It was reported that the drugs can be loaded into the cavity of HNTs feasibly 22-27, which exhibiting the distinctive features such as sustained release, high bioavailability, well water dispersibility and so on. However, the established drug delivery system frequently encountered some disadvantages containing poor drug loading capacity and “initial burst effect” regarded as a puzzling problem in the diffusion-controlled drug delivery. Surface modification upon HNTs offers a feasible route to make the drug release more controllably

28-31

. The stimuli-responsive delivery systems corresponding to

thermal, pH and et al were also prepared based on the surface modification

32-35

.

Obviously, the selective modification of internal or external surface should be the key step for achieving the smart HNTs-based materials. Takahara and co-workers demonstrated that the introduction of dopamine derivatives can selectively modify the 3



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inner surface of HNTs 36. Lazzara et al developed an available method to optimize the external surface by using triazolium salts

32

. Lvov et al reported that the inorganic

micelle-like architecture can be obtained by using the functionalization of the interiors of HNTs, and the immobilized drug based on these materials can be released controllably

37

. Our group also presented a fascinating approach that the

functionalized HNTs with arylboronic acid on innermost surface exhibited positive response to H2O2 with high selectivity, which might provide potential applications in biomedicine field 38. Hydrogen peroxide (H2O2) is one of the most important reactive oxygen species (ROS) involving a variety of pathological effects in organisms

39-40

. It has been

reported that the aberrant production of H2O2 is connected with various diseases, such as cancer, diabetes and cardiovascular

41-44

. The overexpression of H2O2 in

inflammation recurrence was observed and has drawn tremendous attention in clinical, especially in the stage of wound healing after operation

45-47

. Since arylboronic acid

modified-HNTs exhibited specific H2O2-responsiveness, it spurred us to explore HNTs-based H2O2-responsive drug delivery system that could be employed as topical preparations to prevent inflammation occurring. For this purpose, the 1,4-phenylenebisboronic acid (PA)-modified HNTs (HNTs-BO) and the compressible starch were adopted to construct drug delivery systems. The vicinal diol groups on the compressible starch can react with arylboronic acid unit on HNTs-BO and then form a chemical hydrogel (HCH) 48-49. The B-C bond in the formed hydrogel can be broken in the presence of H2O2 resulting into the degradation of hydrogel. These materials could further be developed as the drug delivery systems via two different approaches. The model drug (pentoxifylline, PTX) was initially loaded into the lumen of HNTs-BO and then used to form hydrogel-based drug delivery system (DLHCH-1) as mentioned above. The other one for making drug-loaded hydrogel (DLHCH-2) was achieved by adding the drug into the reaction medium before gel formation, in which the model drug was dispersed in the networks. Both of them exhibited the H2O2-responsive behaviour. Interestingly, 4


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DLHCH-1 showed a much better inhibition effect on “initial burst effect” than that of DLHCH-2. The “initial burst release” can be seen as an inherent property of diffusion-controlled drug delivery systems. An excessive drug release in the burst phase may be toxic and result in depressed bioavailability 50-51. Thus the “initial burst release” is regarded as a puzzling problem in the diffusion-controlled drug delivery systems. It is full of significance to suppress the “initial burst release” in the field of pharmaceutics.

2. Experimental 2.1. Materials HNTs were obtained from GuangZhouShinshi Metallurgy. and Chemical Company

Ltd

(Guangzhou,

China).

Pentoxifylline

(PTX)

and

1,4-

phenylenebisdiboronic acid (PA) were purchased from Sigma-Aldrich. Compressible starch was kindly provided by Colorcon Co., Ltd. All the organic solvents were distilled through the standard method. Dimethyl sulfoxide (DMSO) was dried and distilled from CaH2 under vacuum. Distilled water was used throughout the study. High-purity argon was used for degassing procedures. 2.2 Preparation 2.2.1 Purification of HNTs HNTs were purified according to our previous work 52. 2.2.2 Preparation of HNTs-BO A mixture of purified HNTs (200 mg) and PA (400 mg) in anhydrous DMSO was carefully degassed. The system was heated at 80 oC for 6 h under stirring. The mixture was cooled to ambient temperature and then washed sequentially by ethyl acetate, methanol and dichloromethane. The residue was collected by centrifugation. After vacuum-drying, the HNTs-BO was obtained as a faint yellow solid.

5



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Scheme 1. The preparation of HNTs-BO by grafting PA to the alumina innermost of HNTs (We have demonstrated in the previous study that the overwhelming majority modification occurred in the inner surface of HNTs 38. The outer alumosilicate sheets of the rolls are not tightly bound and the very last one is often loose. So, this very last sheet has an essential part of alumina surface opened to the bulk. Maybe a slight amount of PA was bound onto the outface of HNTs, which is quite helpful in the gel formation.)

2.2.3 Preparation of HCH Compressible starch (200 mg) was dissolved in 2.0 mL of distilled water at 80 oC. HNTs-BO (100 mg) dispersed in 1.0 mL of distilled water was added carefully into the above aqueous solution. Milky white hydrogel was formed after stirred for about 10 mins. The obtained HCH was stored in ambient temperature before use. (shown in Scheme 2A)

6


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Scheme 2. Preparation of HNTs-based hydrogels. A) The route and mechanism of preparing HCH; B) Preparation of DLHNTs-BO and DLHCH-1 (PTX was loaded in the lumen in DLHNTs-BO and DLHCH-1); C) Preparation of DLHCH-2 (PTX was embedded in the matrix network in DLHCH-2.

2.2.4 Preparation of drug-loaded HNTs-BO (DLHNTs-BO) 200 mg of HNTs-BO was dispersed into a saturated solution of pentoxifylline in DMSO. The suspension was placed in a vacuum chamber at an ultrasound power of 200 W for 5 mins. The product was washed with acetone and then dried in vacuum to give DLHNTs-BO as white powder. (shown in Scheme 2B) 7



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2.2.5 Preparation of DLHCH-1 Compressible starch (200 mg) was dissolved in 2.0 mL of distilled water at 80 oC. 100 mg of DLHNTs-BO dispersed in 1.0 mL of distilled water, and then it was added carefully into the above aqueous solution. Milky white hydrogel was formed after stirred for about 10 minutes. The obtained DLHCH-1 was stored in ambient temperature before use. (shown in Scheme 2B) 2.2.6 Preparation of DLHCH-2 Compressible starch (200 mg) and PTX (10 mg) were dissolved in 2.0 mL of distilled water at 80 oC. 100 mg of HNTs-BO dispersed in 1.0 mL of distilled water, and then it was added carefully into the compressible starch and PTX solution. Milky white hydrogel was formed after stirred for about 10 mins. The prepared drug-loaded DLHCH-2 was stored in ambient temperature before use. (shown in Scheme 2C) 2.3 Drug delivery Drug delivery studies were carried out using a ZRS-8G dissolution tester (Haiyida, China). The paddle rotation speed was set as 50 rpm at 37 ± 0.5 oC. The drug release behaviors of the prepared DLHCH-1 and DLHCH-2 were evaluated by immersing the obtained hydrogels into pH 7.4 phosphate buffer solution (PBS) with H2O2 (0.1 mM) and without H2O2. The samples were filtered through a membrane filter (pore size 0.45 µm) before monitored. The filtrates were directly subjected to UV-visible spectrometer for test. 2.4 Characterizations UV-visible absorption spectra were obtained on a Shimadzu UV-visible spectrometer model UV-2550. TGA was performed on Perkin-Elmer Pyris 6 at a scanning rate of 10 oC/min from 40 to 800 oC under nitrogen. The morphological characterizations were performed by using a Tecnai G2 F20 S-TWIN transmission electron microscope (TEM) with an accelerating voltage of 200 kV and on a JEOL Ltd. JSM-7500F Cryo field emission scanning electron microscopy (SEM). The ﬂuffy samples were carefully put on ﬂat substrates precoated with carbonic glues and then were coated with gold for SEM observations. FTIR spectra were recorded in the 8


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region of 400-4000 cm-1 for each sample on a Varian-640 spectrophotometer. Samples were previously ground and mixed thoroughly with KBr. The 11B and 13C Solid-state NMR spectra were obtained on a Bruker Advance III spectrometer. X-ray photoelectron spectroscopy (XPS) was carried out on a Thermo Scientific ESCALab 250Xi using 200 W monochromated Al Ka radiation. The 500 µm X-ray spot was used for XPS analysis. The base pressure in the analysis chamber was about 3 × 10-10 mbar. Typically, the hydrocarbon C1s line at 284.8 eV from adventitious carbon was used for energy referencing.

3. Results and Discussion HNTs-BO was prepared by treating purified HNTs with excess of PA. DMSO was used as the reaction solvent ensuring the solubility of PA and the dispersibility of the nanotubes. The product was washed by ethyl acetate, methanol and dichloromethane removing the impurities. Solid-state NMR, FTIR, TGA, XPS and TEM analyses were performed to characterize the obtained HNTs-BO. Fig.1A shows the

13

C solid-state NMR spectra of HNTs and HNTs-BO,

respectively. The spectrum of HNTs-BO exhibits a resonance at 135 ppm, while no obvious signal in the region from 250 to 100 ppm is observed for HNTs, which can be attributed to the carbon units in benzene groups matching with reaction mechanism depicted in Scheme 1. The 11B solid-state NMR spectra of HNTs and HNTs-BO were also investigated, shown in Supporting Information. There is no peak in the

11

solid-state NMR spectrum of HNTs, indicating the lack of boron in HNTs. The

11

B B

solid-state NMR spectrum of PA exhibits a broad peak at 22.8 ppm. It should be noted that a broad peak centralizing at 26.5 ppm was emerged for HNTs-BO. The peak shifted to low field from 22.8 to 26.5 ppm. The result implies that the HNTs-BO is not a simple physical mixture, and the PA units are covalently bond to HNTs in HNTs-BO. The XPS spectra (shown in Supporting Information) of HNTs and HNTs-BO show the presence of aluminum (Al 2p and Al 2s), silicon (Si 3p) and oxygen at 74.8, 9



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119.6, 103.2 and 532.2 eV, matches well with the composition of aluminosilicate clay 37

. It should be noted that the slight signal assigned to B 1s can be tracked at 191.5 eV,

suggesting the presence of boron in HNTs-BO.

Figure 1. Characterizations: A)

13

C solid-state NMR spectra of HNTs and HNTs-BO; B) FTIR

spectra of HNTs, HNTs-BO, DLHNTs-BO and HCH.

Fig.1B shows the FTIR spectra of HNTs and HNTs-BO 53. A significant decrease of the peak intensity at 3697 cm-1 is clearly observed, suggesting the consumption of surface hydroxyl groups in lumen during the modification. While the peak at 3626 cm-1 is unchanged because hydroxyl groups lying between the tetrahedral and 10


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octahedral sheets could not react with arylboronic acid. The new peaks centered at 3408, 3228, 3017 and 1414 cm-1 were found for HNTs-BO. The peaks at 3017 cm-1 and 1414 cm-1 belong to C-H stretching and benzene skeleton vibration, respectively. The peaks at 3408 and 3228 cm-1 are assigned to the unreacted -OH groups in PA units. All the other characteristic peaks assigned to HNTs, including Si–O–Si stretching vibration at 1036 cm-1 and the symmetric or perpendicular stretching vibrations of Si–O or Al–O groups less than 1000 cm-1 are kept, indicating the modification was finished selectively on the internal surface of HNTs.

11



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Figure 2. TEM images of HNTs (A), HNTs-BO (B) and DLHNTs-BO (C)

TGA curves of HNTs and HNTs-BO are illustrated in Supporting Information. The slight mass loss before 100 oC can be attributed to the reduction of the adsorbed water on HNTs. The crystal water was removed as the temperature increased from 200 to 400 oC. The condensation reaction between Al-OH groups led to the weight loss from 400 to 600 oC. The quantity of the PA grafted on the nanotube was 12


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evaluated to be ca. 8.9 wt%, which was determined by the residual masses at 800 oC of HNTs-BO and HNTs, respectively. As shown in Figure 2A, the nanotubes of HNTs present cylindrical shaped with an open-ended lumen. As for HNTs-BO (Fig.2B), the similar morphologies are observed, indicating no damage during the modification procedure. The interior cavity of the HNTs-BO becomes less transparent because PA was grafted on the lumen surface of HNTs, and the outmost surface retains smooth. The aforementioned information infers that the majority modification occurred in the inner surface of HNTs, in accordance with our previous finding

38

. In addition, the TEM images

indicate that the outer alumosilicate sheets of the rolls are not tightly bound and the very last one is loose. So, this very last sheet has an essential part of alumina surface opened to the bulk. This part will be also linked with PA units and may be an important factor for future gel formation because it is better accessible. The arylboronic acid groups at the tube opening of HNTs-BO and a small quantity on the very last sheet are capable of reacting with the vicinal diol groups in compressible starch. As suspension of HNTs-BO adding into the aqueous solution of compressible starch, the mixture became stiff rapidly and the self-standing hydrogel was formed within ca. 10 mins as depicted in Fig.3. The obtained hydrogel (HCH) was treated by freeze-drying avoiding shrink-age, and then be characterized by FTIR, and SEM. Fig.1B gives the FTIR spectrum of HCH. The peaks located at 3697, 3626, 1415, 1038 are corresponding to Al-OH groups spread on the lumen surface, Al-OH groups lying between the tetrahedral and octahedral sheets, aromatic rings and Si-O-Si groups in HNTs-BO, respectively. The bands located at 3429 and 2926 cm-1 can be attributed to the -OH and C-H groups in compressible starch. The peak assigned to the B–OH groups is disappeared in the spectrum of HCH, since B-OH groups have been reacted with the vicinal diol groups in compressible starch after the hydrogel formation.

13



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Figure 3. Photographs of the prepared HCH (A: the chemical gel upon addition of HNTs-BO suspension to the solution; B: the colour of the gel is white; C: the size of prepared hydrogel; D) the gel is free standing.)

The microstructure of HCH is characterized by SEM shown in Fig.4. The porous structure is observed at the magnification of 200 times (Fig.4A). The fracture surface of HCH at the magnification of 3×104 times is shown in Fig.4B, and the surface morphology of HCH is given in Fig.4C at 5×104 times. Those two images (Fig.4B and C) confirm the existence of the nanotubes in the as-formed hydrogel.

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Figure 4. SEM images of HCH. (A: the morphology at the magnification of 200 times; B: fracture surface of HCH at the magnification of 3×104 times; C: the surface morphology at the magnification of 5×104 times)

The drug loading procedure was achieved by using two different approaches. For 15



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the DLHCH-1 system, PTX was initially loaded into the lumen of HNTs-BO by a typical vacuum cycling. This cycle was repeated several times in order to get the most optimized loading efficiency. After finished, the HNTs-BO/PTX complex was washed in order to remove the drugs adhered on the outer surface. Additionally, a physical mixture of HNTs-BO and the drug (PM2) was also prepared by simply mixing the two components in a mass ratio of 10:1, where the content of the drug in PM2 is similar to that in HNTs-BO. The XPS spectra of DLHNTs-BO and PM2 were given in the Supporting Information. The wall thickness of HNTs exceeds the penetration depth of XPS. It is difficult to disclose the chemical composition in the lumen

36

. The N 1s

peak at 399.4 eV can be clearly observed in the XPS spectrum of PM2, which is attributed to the nitrogen in the drug. While the signal was not detected in the spectrum of DLHNTs-BO, which indicated that the drug was loaded in the lumen of HNTs rather than adsorbed on the external area. The loading of PTX in the cavity of HNTs-BO was also evidenced by using FTIR (Fig.1B) and TEM observations (Fig.2C). The drug loading amount was evaluated as 8.6% based on the TGA (Supporting Information). DLHCH-2 is prepared simply by dissolving the model drug (PTX) in compressible starch aqueous solution directly and then adding HNTs-BO to achieve the drug-loaded hydrogel.

Figure 5. The H2O2-responsiveness of HCH, DLHCH-1 and DLHCH-2. (A: HCH in a transparent vial; B: HCH immersing in H2O2 aqueous solution on a rotating shaker at 50 rpm for 100 min; C: the gel immersing in aqueous solution on a rotating shaker at 50 rpm for 100 min; D: the drug 16


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release profiles of DLHCH-1 and DLHCH-2)

The B-C linkage in the obtained hydrogels can be degraded into B-OH and C-OH groups in the presence of H2O2, which has been confirmed in our previous study

38

. HCH was sealed in transparent vials and then water or H2O2 solution was

added. The vials were placed on a rotating shaker at 50 rpm aiming to verify the H2O2-responsiveness of HCH. As shown in Fig.5C, the hydrogel immersing in water over 100 min retained the self-standing character, and in contrast the hydrogel gradually dispersed into the H2O2 solution ([H2O2]=0.1 mM) and then gave a uniform suspension after 100 min (shown in Fig.5B). The results demonstrate the H2O2-responsiveness of the hydrogel.

Figure 6. The release mechanism of DLHCH-1

The cumulative release profiles of PTX were performed in PBS solution with and without H2O2 shown in Fig.5D. There is a small amount of PTX was released (90%) of PTX was achieved within 60 min. Moreover, the similarity factor between those two curves was calculated as f2= 52

57

. The result indicated that there was a negligible difference

between the release curves of DLHCH-1 and DLHCH-2 corresponding to the H2O2-responsive character, although a slightly higher release rate was observed for DLHCH-2.

4. Conclusions In summary, HNTs was modified by using 1,4-phenylenebisdiboronic acid via dehydration condensation in which the desirable dispersibility in aqueous solution of obtained HNTs-BO was kept. A feasible approach was given by using HNTs-BO and 18


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compressible starch to construct a hydrogel-based drug delivery system, where HNTs-BO was adopted as drug carrier. The as-formed hydrogel expressed H2O2-responsive character because the B-C linkage was broken in the presence of H2O2, resulting in the degradation of hydrogel. The “initial burst effect” was effectively suppressed for DLHCH-1, which can be owing to that the drug releasing behavior from the hydrogel into the medium was restrained. Our method provides a promising opportunity to design and prepare novel drug delivery systems and extend their potential application in biomedicine field such as topical preparations in the stage of wound healing to prevent inflammation occurring, where H2O2 in inflammation recurrence was adopted as trigger molecule.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant Nos. 21274037, 21304029 and 21474026) and the Outstanding Doctoral Cultivation Project of Hebei University (YB201501).

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The as-formed hydrogel expressed H2O2-responsive character because the B-C linkage were broken in the presence of H2O2 resulting in the degradation of hydrogel. The “initial burst effect” was suppressed for drugloaded HCH since the drug loaded in the nanotubes releasing from the cavities into the medium was restrained effectively. 299x302mm (96 x 96 DPI)


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A Smart H2O2-Responsive Drug Delivery System Made by Halloysite

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