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Dongwei Wu, Wuxiao Ding, Naohiro Kameta

Functionalized organic nanotubes with highly

tunable crosslinking site density for mechanical

enhancement and pH-controlled drug release of

nanocomposite hydrogels

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Wu, D., Ding, W., & Kameta, N. (2021). Functionalized organic nanotubes with highly tunable crosslinking site

density for mechanical enhancement and pH-controlled drug release of nanocomposite hydrogels. In Polymer

Journal (Vol. 54, Issue 1, pp. 67–78). Springer Science and Business Media LLC.

https://doi.org/10.1038/s41428-021-00556-1.

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Functionalized organic nanotubes with highly tunable

crosslinking site density for mechanical enhancement and

pH-controlled drug release of nanocomposite hydrogels

Dongwei Wu,a,b,c Wuxiao Ding,a,* Naohiro Kametaa

a Nanomaterials Research Institute, National Institute of Advanced Industrial Science and Technology,

Tsukuba Central 5, 1-1-1 Higashi, Tsukuba, Ibaraki 305-8565, Japan

b Department of Applied Biochemistry, Institute of Biotechnology, 4/3-2, Technische Universität

Berlin, Gustav-Meyer-Allee 25, 13355 Berlin, Germany

c Department of Materials Science and Engineering, Jinan University, Guangzhou 510632, China

Abstract:

Organic nanotubes (ONTs) have attracted growing attention in biomedical

applications because of their unique inner and outer nanospaces. Here, ONTs were

functionalized and hybridized with poly(ethylene glycol) (PEG) to construct

nanocomposite hydrogels, with the aim of enhancing their mechanical strength and

controlling their release properties. These nanoengineered hydrogels have 4-fold

greater mechanical stiffness than unreinforced hydrogels and show a more stable

network. The effects of ONT concentration and crosslinkable site density on the

hydrogel mechanical properties were systematically assessed. Moreover, the

incorporation of ONTs enabled simple and effective post-loading of the model drug, as

well as a sustained drug release profile from the hydrogels. These results provide a

novel method to generate mechanically enhanced nanocomposite hydrogels with

improved drug delivery in an easy, efficient and tunable manner, and the obtained

nanocomposite hydrogels may have potential applications in drug delivery and other

related bioapplications.

Keywords:

Drug delivery / Nanocomposite / Organic nanotubes / Reinforced hydrogels

1. Introduction

Hydrogels are interpenetrating three-dimensional polymeric networks with a high

water content that can closely mimic native tissue microenvironments1, 2. They have

found significant applications, such as in drug delivery and regenerative medicine, in

recent decades3, 4. However, the use of hydrogels in biomedical fields remain severely

hampered due to their limited mechanical properties and trade-off between drug loading

capacity and sustained release 5-7. Therefore, numerous efforts have been made to

improve the mechanical performances of hydrogels, such as with double-network

hydrogels8, topological hydrogels9 and nanocomposite hydrogels10-12. Notably,

nanocomposite hydrogels incorporating different types of nanostructures have attracted

increasing attention in biomedical fields 13.

Among the various types of nanostructures used in nanocomposite hydrogels,

tubular nanostructures have been demonstrated to address the shortcomings of

conventional hydrogels, as incorporated nanotubes that are either covalently or

noncovalently bound to the hydrogel network can not only mechanically reinforce the

matrix but also lead to improved drug loading and release behaviors. Moreover,

nanotubes, owing to their high aspect ratio and one-dimensional nanospace as a drug

reservoir, endow composite hydrogels with a favorable drug-loading capacity and

stability to effectively control drug release14, 15. For example, it was reported that a

nanohybrid silk hydrogel with single-walled carbon nanotubes showed significantly

enhanced mechanical strength and sustained doxorubicin release16. Ribeiro et al. used

halloysite aluminosilicate nanotubes and gelatin methacryloyl to formulate composite

hydrogels as an injectable drug delivery system for dental infection ablation17. Many

studies have been carried out to investigate the mechanical reinforcement and drug

delivery enhancement properties of nanotube composite hydrogels. However,

controlling the mechanical properties is desirable so that the hydrogel can match the

properties of the target tissue. To this end, currently, most nanocomposite hydrogels

require changing the polymer concentration or quantity of incorporated nanoparticles

that adjust the crosslinking density and mechanical behaviors. Nevertheless, these

changes will also influence the microstructure, drug loading capacity and release

behavior of the composite hydrogels.

In addition to inorganic nanotubes, organic nanotubes (ONTs) commonly self-

assemble from polymers and small amphiphilic molecules, providing an attractive

platform for diverse biomedical applications by utilizing their unique one-dimensional

nanospace and exterior outer surfaces18-22. To explore their biomedical applications,

ONTs have been further fabricated into hydrogels as platforms for DNA delivery and

protein refolding23, 24, utilizing noncovalent bonding with the hydrogel. Herein, we

propose a simple and efficient way to produce crosslinkable ONTs with variable

numbers of crosslinkable sites on the surface, which were further incorporated into

poly(ethylene glycol) dimethylacrylate (PEGDMA) to generate nanocomposite

hydrogels (Fig. 1). It is expected that the mechanical performance of these

nanoengineered hydrogels can be easily tailored by manipulating the crosslinkable site

density on the nanotube surface as well as the concentration of reinforcing nanotubes.

We also predict that the introduction of ONTs can affect the physical stability of the

hybrid hydrogels and drug encapsulation/release profiles. Highly adjustable ONTs can

be used to tune the mechanical properties and drug loading and release capacities of

hydrogels, particularly to decouple their direct association. The method in this study

may have broad utility in the production of various nanoengineered hydrogels for

biomedical applications, such as tissue-engineered scaffolds, drug delivery systems and

wound dressings.

2. Materials and methods

2.1. Materials

Tetradecanedioic acid, methacryloyl chloride, N-Boc-2,2′-

(ethylenedioxy)diethylamine (Boc-PEO2-NH2), and Boc-PEO9-NH2 were purchased

from TCI Co., Ltd. (Tokyo, Japan). 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-

methylmorpholinium chloride (DMT-MM), 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide hydrochloride (WSC·HCl), N-

hydroxysuccinimide (HOSu), 2,2'-azobis[2-(2-imidazolin-2-yl)propane]

dihydrochloride (AIPH), and other organic solvents were provided by FUJIFILM Wako

Pure Chemicals Co., Ltd. (Tokyo, Japan). Poly(ethylene glycol) dimethylacrylates

(PEGDMAs; average Mn 750) were purchased from Sigma–Aldrich (MO, USA).

2.2. Synthesis of PEGDMA6000, the ONT-forming lipid, and

crosslinkable lipids

PEGDMA with a molecular weight of 6000 was synthesized according to Son’s

paper25. The ONT-forming lipid was synthesized as reported previously26. The

crosslinkable lipids, denoted C2-lipid and C9-lipid, were synthesized according to

Scheme 1.

Tetradecanedioic acid (400 mg, 1.55 mmol) was dissolved in 20 mL of methanol,

to which DMT-MM (250 mg, 0.77 mmol) and NH2-Gly4-OMe (202 mg, 0.77 mmol)

were sequentially added. Target compound lipid 1 formed and precipitated quickly due

to its low solubility in methanol. After stirring at room temperature overnight, the turbid

dispersion was filtered and washed with methanol to obtain lipid 1 (363 mg, yield 94%).

Boc-PEO2-NH2 (200 mg, 0.8 mmol) and triethylamine (120 µL, 0.86 mmol) were

added to 10 mL of dichloromethane. The solution was chilled in an ice water bath, to

which methacryloyl chloride (86 µL, 0.86 mmol) was added dropwise. The solution

stirred for 1 h in an ice water bath and then 1 h at room temperature. The

dichloromethane was removed by vacuum evaporation and further dried overnight

under vacuum. Next, 2 N HCl methanolic solution was added to the above intermediate

to remove the Boc protecting group to obtain lipid 2.

Lipid 1 (363 mg, 0.72 mmol) was dissolved in 30 mL of dimethyl sulfoxide

(DMSO), into which WSC·HCl (200 mg, 1 mmol) and HOSu (102 mg, 0.88 mmol)

were added followed by stirring at RT for 2 h. Lipid 2 (203 mg, 0.8 mmol) and

triethylamine (120 µL, 0.86 mmol) were then added to the above solution to react

overnight at room temperature. DMSO was removed by freeze-drying, and the solid

was redispersed in 90 mL of ethanol-water solution (2/1 volume ratio). The dispersion

was filtered and dried to obtain the methyl ester form of C2-lipid (445 mg, 89% yield).

After hydrolysis with 1 N NaOH and pH adjustment with 1 N HCl, 351 mg of C2-lipid

was collected (81% total yield). C9-lipid, which has a long spacer moiety, was

synthesized according to the same protocol by using Boc-PEO9-NH2 as a starting

material, giving a total yield of 79%.

H-NMR of

C2-lipid

[dimethyl sulfoxide (DMSO)-d6, 400 MHz]: δ: 1.23 (m, 16H,

–CH2–), 1.47 (m, 4H, –NHCO–CH2–CH2–CH2–), 1.84 (s, 3H, CH3–C(CH2)–CONH–),

2.13 (tt, 4H, –NHCO–CH2–CH2–CH2–), 3.3~3.5 (t, 12H, –CONH–CH2–CH2–O–CH2–

CH2–O–CH2–CH2–NHCO–), 3.73 (qd, 8H, –CONH–CH2–CO–), 5.32 and 5.65 (s, 2H,

CH3–C(CH2)–CONH–), 7.82 and 7.93 (t, 2H, –CONH–CH2–CH2–O–CH2–CH2–O–

CH2–CH2–NHCO–), 8.10 (m, 4H, –CONH–CH2–) ppm. The

H-NMR spectrum of

C9-lipid is quite similar to that of C2-lipid, and the integration of the peak between 3.3

and 3.5 ppm of C9-lipid was 39H.

C-NMR of

C2-lipid

[dimethyl sulfoxide (DMSO)-d6, 400 MHz]: δ: 19.1 (CH3–

C(CH2)–CONH–), 25.6~29.5 (–CH2–CH2–CH2–), 35.6 and 35.8 (–NHCO–CH2–CH2–

CH2–), 38.9 and 39.4 (–CONH–CH2–CH2–O–CH2–CH2–O–CH2–CH2–NHCO–),

41.1~42.5 (–CONH–CH2–CO–), 69.7 and 70.0 (–CONH–CH2–CH2–O–CH2–CH2–O–

CH2–CH2–NHCO–), 119.5 (CH3–C(CH2)–CONH–), 140.3 (CH3–C(CH2)–CONH–),

168.0 (CH3–C(CH2)–CONH–), 171.6 (–CONH–CH2–CO–), 173.2 and 175.0 (–

NHCO–CH2–CH2–CH2–), 174.2 (–CONH–CH2–COOH) ppm. The

C-NMR

spectrum of

C9-lipid is quite similar to that of C2-lipid, and the peak at 70.0 ppm is

much stronger for C9-lipid.

Elemental analysis: anal. calcd for C32H56N6O10 (C2-lipid): C, 56.12; H, 8.24; N,

12.27; O, 23.36; found: C, 56.85; H, 8. 41; N, 12.37; O, 22.37. Anal. calcd for

C46H84N6O17 (C9-lipid): C, 55.63; H, 8.53; N, 8.46; O, 27.38; found: C, 56.15; H, 8.70;

N, 8.57; O, 26.58.

1H-NMR, 13C-NMR and FT-IR spectra of C2-lipid and C9-lipid are presented in

Figs. S1-S3 in the supporting information (SI).

2.3. Assembly of crosslinkable ONTs

Crosslinkable nanotubes (C-ONT) were constructed by the coassembly the of ONT-

forming lipid and crosslinkable lipids by pH adjustment, as reported for the bare ONTs.

Briefly, 60 mg of ONT-forming lipid and different amounts of crosslinkable C2-lipid

or C9-lipid (0.05, 0.5, and 5 mol% ONT-forming lipid) were dispersed in 20 mL of

Milli-Q water, followed by the addition of 1 N NaOH solution to yield a clear lipid

solution. Next, a 1 N HCl solution was used to adjust the pH to 4~5 to form ONT

dispersions. The ONTs were collected by filtration through a polycarbonate membrane

(100 nm pore size) and redispersed in Milli-Q water at a concentration of 20 mg/mL

(2%, calculated from the original ONT-forming lipid). ONTs functionalized with 5 mol%

C2-lipid and C9-lipid were named C2-ONT and C9-ONT, respectively. C2-ONT and

C9-ONT are collectively named C-ONT. The nanotube morphologies of these C-ONTs

were confirmed by scanning transmission electron microscopy (STEM) with a Hitachi

S-4800 microscope (Tokyo, Japan) after negative staining with phosphotungstate

solution.

2.4. Preparation of the nanocomposite hydrogels

Composite hydrogels with different formulations were prepared by mixing

PEGDMA aqueous solution and ONT solution at various ratios with 0.5% (w/v)

photoinitiator AIPH at room temperature. The mixed solutions were then exposed to

365 nm UV light (50 mW/cm2) for 3 min to form composite hydrogels; the distance

between each sample and UV lamp was 10 cm. Pure PEGDMA hydrogels were also

prepared by photopolymerization following similar processes. The nanocomposite

hydrogels composed of 10% PEGDMA and 1% C-ONT were named C2-ONT-PEG750,

C9-ONT-PEG750, C2-ONT-PEG6000, and C9-ONT-PEG6000, respectively.

2.5. Rheological testing

A rheological study was carried out with a rotational rheometer (DHR, TA, USA)

equipped with a UV crosslinking instrument (OmniCure S2000, MA, USA). In this

experiment, a parallel plate 20 mm in diameter was used with a gap of 1 mm. To

establish the linear viscoelastic region, the storage modulus (G′) and loss modulus (G′′)

were measured as a function of strain (0.1%~30%) at a frequency of 1 Hz at 25 °C. The

mechanical strength of the hydrogels was also determined. Once the linear viscoelastic

region was established, a dynamic frequency sweep was carried out to record the

viscoelastic parameter change in the frequency range from 0.1 to 100 Hz. The loss

factor tan δ (G′′/G′) was calculated based on the frequency sweep data. In addition, the

crosslinking processes of different samples were monitored under UV irradiation

(50 mW/cm2) for 3 min, during which the moduli of the hydrogels were recorded.

2.6. Swelling ratio measurements

The equilibrium swelling ratios were measured using a gravimetric method. Before

the swelling experiment, the samples were weighed to record their original mass as W0.

The hydrogel samples were swollen then in aqueous phosphate-buffered saline (PBS)

solution at pH = 7.4 for 24 h, and the weight was recorded as Wswell after blotting the

excess surface water with a Kimwipe. The samples were then placed in vacuum at 60 °C

for 48 h to obtain the dry weight (Wdry). The equilibrium swelling ratio and solution

uptake were calculated according to the following formulas, respectively27:

𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆𝑆 𝑟𝑟𝑟𝑟𝑟𝑟𝑆𝑆𝑟𝑟 =𝑊𝑊𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝑊𝑊

𝑊𝑊

× 100% (1)

𝑆𝑆𝑟𝑟𝑆𝑆𝑆𝑆𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆 𝑆𝑆𝑢𝑢𝑟𝑟𝑟𝑟𝑢𝑢𝑆𝑆 =𝑊𝑊𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠𝑠−𝑊𝑊𝑑𝑑𝑑𝑑𝑑𝑑

𝑊𝑊𝑑𝑑𝑑𝑑𝑑𝑑

(2)

2.7. Drug loading and effects of pH adjustment on drug encapsulation

Methylene blue is a water-soluble blue dye with a strong absorbance peak at 664

nm. It was used as the model drug in this study. Methylene blue was dissolved in

deionized water to prepare drug stock solutions at concentrations of 0.1 mg/mL, 0.5

mg/mL, 1 mg/mL, 2 mg/mL and 5 mg/mL with or without pH adjustment to 8.0 with

NaOH solution. Hydrogel samples of the same weight were immersed in 1.0 mL of

methylene blue solution at room temperature for 48 h in the dark. Subsequently, the

supernatant was collected and the absorbance was measured by UV-Vis spectroscopy

at 664 nm (Shimadzu, Japan). The drug-loaded hydrogel samples were then vacuum-

dried at 60 °C for 48 h to remove any residual moisture and weighed. The loading

capacity and encapsulation efficiency were determined by the following equations28:

𝐸𝐸𝑆𝑆𝐸𝐸𝑟𝑟𝑢𝑢𝐸𝐸𝑆𝑆𝑆𝑆𝑟𝑟𝑟𝑟𝑆𝑆𝑟𝑟𝑆𝑆 𝑆𝑆𝑒𝑒𝑒𝑒𝑆𝑆𝐸𝐸𝑆𝑆𝑆𝑆𝑆𝑆𝐸𝐸𝑒𝑒 =𝑊𝑊𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑠𝑠−𝑊𝑊𝑑𝑑𝑠𝑠𝑟𝑟𝑡𝑡𝑟𝑟𝑟𝑟

𝑊𝑊𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑠𝑠

× 100% (3)

𝐿𝐿𝑟𝑟𝑟𝑟𝐿𝐿𝑆𝑆𝑆𝑆𝑆𝑆 𝐸𝐸𝑟𝑟𝑢𝑢𝑟𝑟𝐸𝐸𝑆𝑆𝑟𝑟𝑒𝑒 =𝑊𝑊𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑡𝑠𝑠−𝑊𝑊𝑑𝑑𝑠𝑠𝑟𝑟𝑡𝑡𝑟𝑟𝑟𝑟

𝑊𝑊𝑠𝑠𝑡𝑡𝑟𝑟𝑠𝑠𝑠𝑠𝑠𝑠

× 100% (4)

where Wtotal and Wremain represent the initial amount of methylene blue and the

remaining amount of methylene blue in the supernatant, respectively, and Wsample refers

to the weight of the dry drug-loaded hydrogels.

2.8. In vitro drug release

The release of methylene blue from the hydrogels was carried out in PBS at pH 7.4

or pH 5.5 at 37 °C with stirring. Drug-loaded hydrogels were placed in 5 mL of PBS,

and 1 mL of release solution was withdrawn at appropriate time intervals for UV-Vis

analysis at 664 nm. Subsequently, 1 mL of fresh PBS was added back to the tube to

maintain the same volume. All release experiments were performed in triplicate, and

the cumulative release of methylene blue was calculated and averaged.

3. Results and discussion

3.1. Characterization of the coassembled ONTs

The crosslinkable lipids and ONT-forming lipids were designed to have the same

hydrophobic and tetraglycine segments; therefore, pH adjustment enables the

coassembly of these lipids into crosslinkable ONTs via hydrophobic interactions and

polyglycine hydrogen bonding28-30. Both C2-ONT and C9-ONT showed tubular

morphologies and dimensions very similar to those of bare ONTs (Fig. 2), namely, outer

diameters of ~13 nm, inner diameters of ~ 7 nm, and a single-walled structure. We

previously established the construction of PEG2000-modified ONTs, in which the molar

ratio of PEG2000-lipid can be as high as 20%, and PEGylation resulted in shortening of

the ONTs29. Therefore, at molar ratios of 5%, the C2-lipid and C9-lipid present should

be completely incorporated into the corresponding ONTs without phase separation.

Compared to the bare ONTs, the improved transparency of the aqueous solutions of C2-

ONT and C9-ONT supported successful ONT functionalization. Incorporation of 5%

C2-lipid and C9-lipid did not significantly change the nanotube lengths, which were in

the range of 200 nm to 610 nm (Fig. S4).

3.2. Crosslinking of the ONTs and formation of the composite hydrogels

The stability of nanocomposite materials is a key issue for nanocomposite

hydrogels. First, we determined the stability of the ONTs after self-crosslinking under

aqueous conditions. As shown in Fig. 3a, after crosslinking, C2-ONT maintained its

tubular structure, and an increase in the solution turbidity also suggested the

crosslinking of C2-ONT. The degree of crosslinking was as low as 2.2% according to

1H-NMR measurements (Fig. S5), and it was supposed that the rigidity of the ONTs

could have prevented further crosslinking once the ONTs were partly entangled.

PEG was selected as the base polymer to prepare the hydrogels, and the flexible and

linear structure was advantageous in terms of accessibility to the surface of stiff ONTs

and the formation of homogenous nanocomposite hydrogels, allowing us to study the

effects of the ONTs in the hydrogels. In Fig. 3b, crosslinkable C2-ONT formed the C2-

ONT-PEG750 nanocomposite hydrogel after UV irradiation. Some filament structures,

which were designed to be C2-ONT covered with PEG750, were observed. In addition,

the storage modulus and loss modulus of the C2-ONT-PEG750 hydrogel were

monitored during UV irradiation, which started to rapidly rise from 12 s and reached a

plateau of over 104 Pa at approximately 60 s. This indicated sufficient crosslinking of

the component and the formation of a mechanically stable hydrogel after 1 min of UV

radiation (Fig. 3c). Therefore, the samples in the following experiments were irradiated

for 3 min to achieve sufficient crosslinking unless otherwise mentioned.

3.3. Hydrogels reinforced with crosslinkable nanotubes

To investigate the effect of ONTs on the mechanical properties of the hydrogels, a

rheometer was used for evaluation after UV crosslinking. As expected, the hydrogel

supplemented with bare ONTs exhibited a storage modulus very similar to that of the

pure PEG hydrogel, which was approximately 5 kPa (Fig. 4a, b). The slight

improvement could be due to the bare ONTs acting as a physical filler in the hydrogel

network. In contrast, the hydrogel with C2-ONT showed significant reinforcement,

reaching a storage modulus of 20 kPa, which was 4 times greater than that of the pure

PEG hydrogel. This result indicates the formation of a covalently crosslinked

nanocomposite hydrogel with a more robust network. For the C9-ONT-incorporated

hydrogel, the storage modulus (approximately 15 kPa) was slightly lower than that of

the hydrogel with C2-ONT but exceeded the value of the pure PEG hydrogel by far.

This might be due to the longer PEO9 segments in the functionalized lipids, which

provide more flexibility to the crosslinked network. To verify this hypothesis, PEG6000

was used to fabricate nanocomposite hydrogels. The rheological tests showed that

PEG6000 displayed the same trend of mechanical reinforcement as PEG750 (Fig. S6).

However, the hydrogels enhanced with C2-ONT and C9-ONT were measured to have

very similar moduli. Thus, the much longer PEG chains on PEG6000 almost

counterbalance the influence of the PEO segments in these functionalized lipids.

The viscoelastic properties of the hydrogels were further studied by frequency

sweep. Fig. 4c reveals that the C-ONT-reinforced hydrogels were much stiffer and

stable than the pure PEG hydrogels or hydrogels with bare ONTs. Across the whole

range of applied frequencies (0.1~100 Hz), all of the hydrogels showed gel-like

behaviors with G′ dominance. However, Fig. 4d shows that the C-ONT composite

hydrogels had a lower and more stable loss factor (tan δ) within the measured frequency

range, suggesting that C-ONT enabled the hydrogel to form a stable network with a

response that was more elastic-like than viscous. In addition, the highest loss factor was

observed in the hydrogel with the bare ONTs due to the additional friction between the

PEG segments and infilled nanotubes in addition to the intermolecular friction of the

PEG chains31, 32.

3.4. Influence of nanotube concentration

To further investigate the effects of crosslinkable ONTs on the mechanical

performance of the nanocomposites, reinforced hydrogels were prepared with 10%

PEG750 and C2-ONT at different compositions of 0.2%, 0.5% and 1%. The

incorporation of a larger amount of C2-ONT resulted in improved stability of the

enhanced hydrogel network, as determined from strain sweep and frequency sweep

testing. Fig. 5 shows that when incorporating 0.2%, 0.5%, and 1% C2-ONT, the

nanocomposite hydrogel had an increasing storage modulus, the value of which rose

significantly from 7.5 kPa to 12.5 kPa and further to approximately 20 kPa. The

frequency sweep test indicated that all of the nanocomposites had a very stable

crosslinking network, as they had very stable storage modulus values, which were much

higher than those of the loss modulus. When monitoring the gelation process under UV

radiation, it was observed that nanocomposites with a higher concentration of C2-ONT

showed a faster increase in their storage modulus and higher values. A larger content of

C2-ONT provided a higher density of functionalized lipids with crosslinkable sites,

leading to a higher probability of crosslinking.

3.5. Effects of crosslinkable site density on the nanotubes

We also tried to control the number of crosslinkable sites on the surfaces of the

nanotubes. Crosslinkable ONT was formed by the coassembly of the ONT-forming

lipid and crosslinkable lipids, the ratio of which can be easily controlled. Consequently,

we prepared three different C-ONTs with diverse densities of 0.05%, 0.5% and 5%

crosslinkable lipids and evaluated the effects of the number of crosslinkable sites on the

mechanical properties of C2-ONT-PEG750.

The strain sweep data showed that the storage modulus of the nanocomposite

hydrogels increased as they contained C2-ONT with a higher density of crosslinkable

sites. Compared to the hydrogel with bare ONTs with no crosslinkable sites, the

nanocomposite hydrogel with 0.05% crosslinkable sites had a 50% higher storage

modulus with a value greater than 7.5 kPa. This result indicates that the crosslinkable

lipids on the nanotubes led to an apparent reinforcement of the hydrogel network even

at a very low density. When the crosslinkable site density increased to 0.5%, the

enhancement from C2-ONT became much more pronounced as the storage modulus of

the nanocomposite increased to approximately 17 kPa, which is more than 3 times that

of the hydrogel with the bare ONTs. As the crosslinkable sites increased further to 5%,

the storage modulus of the nanocomposite hydrogel improved slightly. This indicates

that having 5% crosslinkable sites was likely sufficient for the hydrogel crosslinking

reaction. This might be because a higher density of crosslinkable sites on the surface of

the nanotubes can increase the reaction probability, but an overly high density causes

increased steric hindrance33. This theory can be verified by monitoring the change in

the modulus of the precursor under UV radiation. C2-ONT-PEG with 5% crosslinkable

sites showed the fastest increase in the storage modulus among the groups, but the rate

slowed down and reached a rate that was close to that with 0.5% crosslinkable sites

(Fig. 6d).

In addition, the nanocomposite hydrogels were evaluated by frequency sweeps from

0.1 to 100 Hz. C2-ONT hydrogels with 0.5% and 5% crosslinkable sites were both very

stable within the testing range, and they also had very similar storage moduli (Fig. 6c),

which is consistent with the strain sweep results. However, the hydrogels with bare

ONTs or C2-ONT with 0.05% crosslinkable sites, the storage modulus decreased

slightly at a high shearing frequency, whereas a boost in the loss modulus was observed

for the hydrogels with bare ONTs. This indicates that PEG hydrogels with bare ONTs

or C2-ONT with 0.05% crosslinkable sites was not robust under high-frequency shear,

in particular the former, which had the possibility of destruction. Therefore, C-ONT

was demonstrated to affect the mechanical performance of the composite hydrogels,

and the crosslinkable site density on the nanotube surface was able to tune the

crosslinking network.

3.6. Swelling behaviors and microstructures of the nanocomposite

hydrogels

With the understanding of how the ONTs affect the nanocomposite mechanical

properties, we next investigated the swelling behaviors of the hydrogels with different

kinds of ONTs. PBS mimics physiological conditions, and thus a swelling study with

both PEG750 and PEG6000 incorporated with different kinds of ONTs was carried out

in PBS. The swelling ratio and solution uptake data are presented in Fig. 7a-b, d-e.

PEG750-based hydrogels had negative swelling ratios, while PEG6000-based

hydrogels had positive swelling ratios. PEG chain segments were strongly hydrophilic

due to the ether oxygen, and PEG with longer molecular chains has a larger ratio of

ether oxygens to hydrocarbons, which can increase the hydrophobicity. Moreover,

PEG750 has a much shorter chain length and much higher crosslinking density in the

hydrogel network, which can keep the hydrogel contracted and lead to mass loss in PBS.

Also, in Fig. 7c, f, the four PEG750-based hydrogels exhibited a very dense structure,

as observed by SEM, but the PEG6000-based hydrogels were porous. Notably, there

was no observable change found in the microstructures of the nanocomposites

compared to the basic PEG hydrogels. Fig. 7a shows that the ONT-PEG750 and C2-

ONT-PEG750 hydrogels had swelling ratios that were slightly higher than that of the

pure PEG750 hydrogel, but there was no significant difference. Compared to PEG

molecules, the nanotubes are more rigid, serving as stiff fillers in the hydrogel and

preventing further shrinkage of the matrix. However, a lower swelling ratio was found

for the C9-ONT-PEG750 hydrogel in comparison with PEG750. This may be because

the C9-ONT-reinforced hydrogel had a higher crosslinking density and the longer PEO9

segment on the nanotube surface provided more flexibility in the network. Therefore,

this hydrogel was more prone to contract and lost more water while in the buffer

solution.

Similarly, the C9-ONT-PEG6000 hydrogel had the lowest swelling ratio among the

four PEG6000-based hydrogels. The C2-ONT-PEG6000 hydrogel had a value higher

than that of the C9-ONT-PEG6000 hydrogel and lower than that of the PEG6000

hydrogel. Because C2-ONT can be crosslinked with PEG but has reduced reactivity

compared with C9-ONT, the crosslinkable sites located at the end of the longer flexible

PEO chain had a higher probability of reaction. Interestingly, the ONT-PEG6000

hydrogel had the highest swelling ratio among the hydrogels, suggesting that this

hydrogel still had a crosslinking network even with the lowest density. This was due to

the incorporation of bare ONT, which may block the reaction between the acrylic

groups on PEG to a certain extent34.

Moreover, the PEG750-based hydrogels showed solution uptake degrees of

approximately 7, while PEG6000-based hydrogels had much higher values greater than

20. However, hydrogels with the same base exhibited very similar solution uptake

characteristics. This result suggests that the introduction of nanotubes did not change

the water uptake ability of the hydrogels, which is one of the best features of hydrogel

materials.

3.7. Encapsulation efficiency and loading capacity of the hydrogels

Generally, nanotube materials possess an exceptionally high drug loading capacity

due to their high surface area and the possibility of incorporating additional guests into

their inner cavity. Here, methylene blue was used as a model drug to investigate the

encapsulation efficiency and loading capacity of the nanocomposite hydrogels.

Hydrogels of the same weight without or with crosslinkable nanotubes were placed into

0.1 mg/mL methylene blue aqueous solutions. Only 40% of the drug was encapsulated

into pure PEG hydrogels (Fig. 8a). In contrast, the hydrogel incorporated with C2-ONT

showed a much higher encapsulation efficiency, nearly 90%, which was more than 2

times greater than that of the pure PEG hydrogel. An even higher encapsulation

efficiency of approximately 95% was reached when the drug solution was adjusted to

pH ~ 8, ionizing the carboxyl groups to attract positively charged drug guest molecules,

including methylene blue. The encapsulation efficiency of the C2-ONT-PEG750

hydrogel was also measured in different concentrations of methylene blue solutions

with pH adjustment. Notably, the efficiency decreased with increasing concentration,

but was still greater than 40% in 5 mg/mL methylene blue solution.

The loading capacity of the hydrogels is presented in Fig. 8c and 8d. Similar to the

encapsulation efficiency results, the nanocomposite hydrogels had superior loading

capacity compared with the pure PEG hydrogels. Methylene blue was carried in the

nanotubes via ionic interactions, and the carrying capacity of the hydrogel was

significantly affected by the initial methylene blue concentration. Although a higher

drug concentration can decrease the encapsulation efficiency, it prompts a larger

loading capacity. Methylene blue loading improved dramatically from 0.05% to 10%

by increasing the initial methylene blue concentration from 0.01 to 5 mg/mL. These

data conclusively demonstrate that the addition of C2-ONT greatly enhanced the drug-

carrying efficiency and drug loading capacity of the composite hydrogels.

3.8. Drug release from the hydrogels

The drug release behaviors of the hydrogels were also studied in PBS, which

simulates body fluid, using methylene blue as the model drug. Both the release rate and

cumulative release percentage from the pure PEG750 hydrogel were higher than those

of the nanocomposite hydrogel (Fig. 9). The PEG750 hydrogel had a very similar drug

release profiles at different pH values during the testing period, with an initial burst

release of approximately 45% at 30 min followed by a plateau of approximately 99%

after 24 h. This behavior is because the PEG hydrogel network did not have sufficient

binding force with methylene blue, and therefore, the drug diffused quickly from the

hydrogel into the solution. However, the nanocomposite hydrogel released only 10% of

the loaded methylene blue within the first 30 min, which is less than one-quarter that

released from the PEG750 hydrogel. It was thus suggested that most of the methylene

blue was bound in the composite, and definitely within the incorporated nanotubes. This

is consistent with the drug loading test results.

The release from the C2-ONT-PEG750 hydrogel was much smoother and slower

than that from the PEG750 hydrogel over 24 h. For the same nanocomposite hydrogel,

there was also a notable difference between the release at pH = 7.4 and pH = 5.5. As

shown in Fig. 9a, methylene blue was released more quickly in acidic solution over the

first 12 h, more than 10% greater than that in pH 7.4 PBS. After 24 h, the release reached

a nearly constant value of 42% for the composite hydrogel in neutral PBS. However,

the composite hydrogel at pH 5.5 continued to release methylene blue at a slow pace

for 6 days, accounting for 73% released at the end of the experimental period. Therefore,

the incorporation of nanotubes significantly improved the drug loading capacity of the

hydrogels while also significantly prolonging the release profile via a sustained process

and pH-responsive behavior.

In conclusion, we successfully prepared functionalized organic nanotubes for the

fabrication of mechanically enhanced nanocomposites based on PEG hydrogels. The

crosslinkable sites on the nanotube surface can be easily adjusted to alter the density

and length via a facile assembly process. The incorporation of organic nanotubes

resulted in an increase in mechanical stiffness by more than 4-fold compared with that

of the basic PEG hydrogels, and this stiffness can be easily tailored. Interestingly, the

microstructures of the hydrogels were maintained. This is because these functionalized

nanotubes provide different numbers of active sites distributed within the network

acting as crosslinking epicenters. Extraordinarily, these nanoengineered hydrogels also

showed significant improvements in the drug loading capacity and drug release profile,

favorable behaviors that can be maintained when tuning the mechanical properties by

adjusting the incorporated nanotubes. This method can be very useful for engineering

various hydrogels with appropriate mechanical properties and drug

encapsulation/release performance in biomedical fields, including tissue-engineering

scaffolds, stimuli-responsive devices, controlled drug delivery systems and wound

dressings.

Acknowledgments

This work was supported by a KAKENHI from the Japan Society for the Promotion

of Science (grant no. JP18K09469).

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Figures

Fig. 1 Schematic process for the construction of the ONT nanocomposite PEG

hydrogels.

Fig. 2 STEM images of a) bare ONTs, b) C2-ONT, and c) C9-ONT. d) Dimensions

of the ONTs. The insets are the ONTs in aqueous solutions (2%), and the ONTs are

illustrated in the figures (red dots indicate the crosslinkable methylacrylate groups).

Fig. 3 a) STEM image of C2-ONT after UV crosslinking (inset is the crosslinked

C2-ONT aqueous dispersion). b) SEM image of the C2-ONT-PEG750 composite

hydrogel (inset is the hydrogel image); white arrows indicate filament structures of the

hydrogel. c) Time-dependent change in storage modulus (G′) and loss modulus (G′′) of

the C2-ONT-PEG750 composite hydrogel under UV radiation.

Fig. 4 Effects of different nanotubes on the mechanical properties of covalently

crosslinked hydrogels (based on PEG750). a, b) Strain sweep results; c, d) frequency

sweep results.

Fig. 5 Rheological properties of the nanocomposite hydrogels (based on PEG750)

incorporated with different contents of C2-ONT. a) Strain sweep of the hydrogels. b)

Storage moduli of the hydrogels. c) Frequency sweep of the hydrogels. d) Changes in

the storage modulus during crosslinking.

Fig. 6 Rheological properties of the nanocomposite PEG750 hydrogels incorporated

with bare ONT or C2-ONT containing different densities of crosslinkable lipids. a)

Hydrogel strain sweep results. b) Storage moduli of the hydrogels. c) Hydrogel

frequency sweep result. d) Change in the storage modulus of the hydrogels during

crosslinking.

Fig. 7 Swelling ratio, solution uptake and microstructures of the a, b and c) PEG750

and d, e and f) PEG6000 nanocomposite hydrogels. All composite hydrogels were

incorporated with 1% nanotubes.

Fig. 8 Hydrogel a) encapsulation efficiency in 0.1 mg/mL methylene blue solution

and c) loading capacity in 1 mg/mL methylene blue solution. b) Encapsulation

efficiency and d) loading capacity of the C2-ONT-PEG750 hydrogels in pH-adjusted

methylene blue solutions at various concentrations.

Fig. 9 Methylene blue release from PEG750 hydrogels and C2-ONT-PEG750

hydrogels at diverse pH values during the periods of a) 0 to 12 h and b) 0 to 144 h.

Scheme 1. Synthesis of crosslinkable C2-lipid and C9-lipid, which contain a short

PEO2 and long PEO9 spacer, respectively.