Photocontrollable Intermittent Release of Doxorubicin Hydrochloride

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Photo-controllable intermittent release of doxorubicin hydrochloride from liposomes embedded by azobenzene-contained glycolipid Danyang Liu, Sijia Wang, Shouhong Xu, and Honglai Liu Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b03051 • Publication Date (Web): 26 Sep 2016 Downloaded from http://pubs.acs.org on October 5, 2016

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Photo-controllable intermittent release of doxorubicin hydrochloride from liposomes embedded by azobenzenecontained glycolipid Danyang Liu, Sijia Wang, Shouhong Xu* and Honglai Liu

Key Laboratory for Advanced Materials & Institute of Fine Chemicals, School of Chemistry and Molecular Engineering, East China University of Science and Technology, 130 Meilong Rd, Shanghai 200237, PR China; Fax: 8021 64252921; Tel: 8021 64251942; E-mail: [email protected] (S. Xu)

Supporting Information

Abstract Azobenzene-contained glycolipids GlyAzoCns, newly structured azobenzene derivatives, which have an azobenzene moiety between the galactosyl and carbon chains of various sizes, have been synthesized. The GlyAzoCns undergo reversible photo-induced isomerization in both ethanol solution (free state) and liposomal bilayer (restricted state) upon irradiation with UV and Vis-light alternately. The drug release of Liposome@Gly induced by isomerization was found to be an instantaneous behavior. The photo-induced control of DOX release from liposome was investigated in various modes. The Liposome@Glys have been found to keep the entrapped DOX stably in dark with less than 10 % leakage in 10 h but release nearly 100 % of cargos instantaneously with UV-irradiation. Molecular structure of GlyAzoCns and property of liposomal bilayer were considered as important factors influencing drug release. Among the synthesized GlyAzoCns, GlyAzoC7 showed to be the most efficient photosensitive actuator for controlling drug release. A lower proportion of cholesterol in Liposome@Glys was conducive to promote the release amount. Results indicated that the synthesized GlyAzoCns could act as a role of smart actuators in the liposome bilayer and control the drug to release temporarily and quantitatively. Keyword: azobenzene, photoisomerization, UV-sensitive liposome, intermittent release.

Introduction Since light irradiation has the advantages of not only easy to control but also the non-pollution and high efficiency, 1-3various kinds of photoresponsive materials are developed for releasing drug at fixing time/spot, assessing dynamics of biological processes and visualizing the subtleties of biological structures.4 Azobenzene is mainly in the trans configuration and completes reversible trans-cis transition upon UV-irradiation. Recent years, azobenzene and its derivatives are attracting more and more attentions for designing and preparing photo-controlled materials, such as intelligent soft materials (gel, for example) or specific release systems. Tao Yi et al prepared gels functionalized by azobenzene moieties for photo-responsibility, which were found to promote

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the gel-sol transition upon UV/Vis light irradiation due to their hydrophobicity in trans-form and hydrophilicity in cis-form.5-7 Researches about azobenzene linked to cholesterol8, 9 have been reported to obtain the stable membrane and multi-pulsatile release. Besides, giant unilamellar vesicles (GUVs; 5–200 mm diameter) embedded with azobenzene derivative were used to observe and understand the biomimetic cellular reactions, such as biomembrane permeabilization, membrane fusion, etc. 10-16 Morgan et al prepared liposomes containing acyl chains incorporating azobenzene chromophores as potential “caging” agents for fast release.17 The above works have used azobenzene derivatives for preparing various smart materials, even there are some studies on UV sensitive liposomes, but few of them have clarified the detail process and mechanism of the isomerization behavior at 2-dimension interface. Liposomes are the indispensable vehicles in clinical use of gene therapy and immunotherapy. In this paper, we designed and synthesized newly structured glycolipids, which have an azobenzene moiety between the galactosyl and carbon chains of various sizes, for preparing UV-sensitive liposome. The galactosyl was selected as a hydrophilic head for the purpose of tumor cell target and delivery18. The synthesized azobenzene-galactolipid GlyAzoCns will be embedded into HSPC/DSPG/Chol liposome (Liposome@Gly). Liposomes composed of HSPC were reported to have very good stability and very low leakage (less than 50 % of entrapped molecules at 37 ℃ for 4weeks).19, 20 These liposomes always have a problem of no release when delivered to the morbid sites. The GlyAzoCns in liposome bilayer are expected to act as a role of UV-sensitive switch to achieve multimode releases. The UV sensitivity and photoisomerization behaviors of the GlyAzoCns have been investigated in both organic solvent and/or in liposome bilayer.21 The effects of liposomal composition and GlyAzoCns molar structure on drug release have been discussed for understanding the mechanism of UV-induced drug release behavior. Especially, the behavior of GlyAzoCns isomerization at interface has been emphasized for understanding its influence on biomembrane property and function.

Experimental Section Materials. 1, 2-distearoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DSPG) was purchased from Avanti Polar Lipid (USA), L-α-phosphatidylcholine, hydrogenated (Soy) (HSPC) and cholesterol (Chol) (purity > 98 %) were obtained from Lipoids (Germany). Doxorubicin hydrochloride (DOX) and 1,6-diphenyl-1,3,5-hexatriene (DPH) was purchased from J＆K (China). All the chemicals were used without further purification. Synthesis of GlyAzoCns. We designed a series of photo-responsive glycolipid derivative GlyAzoCns. Their chemical structures were given in Scheme S1 with other lipids. Details for synthesis were available in supporting information (Scheme S2, Figure S1-Figure S14). Liposome preparation. The liposomes were prepared using the standard thin-film hydration method. Typically, the phospholipids, cholesterol and GlyAzoCns were dissolved in the mixture of CHCl3 and CH3OH and then transferred into flasks. The thin lipid film formed on the bottom of flask by evaporation at 45 ℃ for 45 min and further dried under vacuum for 1 h at room temperature. For the experiments of photoisomerization, the samples were hydrated at 72 ℃ in a water bath with 10 mM PB (pH 7.40) solution and sonicated for 5 min. The liposomes were then extruded 21


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cycles through 200 nm pore-diameter polycarbonate filters using an extruder (LiposoFast, LF-1, Avestin, Canada). For release and leakage measurements, the thin lipid film was hydrated with 250 mM ammonium sulfate solution and sonicated for 20 min at 72 ℃. The composition of liposomes were shown in Table 1.The final concentration of liposomes were 5 mM (total molar concentration of HSPC, DSPG and Chol). The average sizes and surface charges of liposomes were determined by a Malvern Zetasizer Nano ZS (UK) at 25 ℃. The fluorescence polarizations (P) of liposomes were measured at 37 °C by a fluorophotometer (Perkin Elmer, LS55, USA) using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a fluorescence probe22. Photo-isomerization of GlyAzoCns in ethanol and liposomal bilayer. To determine the UVsensitivity of GlyAzoCns, 0.1 M stocks of GlyAzoCns both in ethanol solution and in liposome bilayer were prepared. The isomerization process was recorded by using a UV-Vis spectrophotometer (UV-2450, Shimadzu, Japan). The GlyAzoCns in ethanol solution or liposome bilayer was irradiated with a 8 W Hg lamp (λ= 365 nm, WFH-204B, He Qi, Shanghai, China) for the trans to cis isomerization and a 60 W Hg lamp (λ= 470 nm, APV-6024, Hai Zhi Ling, Shanghai, China) to relax back. TEM. The liposomes were observed by transmission electron microscope (TEM, JEOL, JEM1400, Japan) before and after irradiation of 365 nm UV light. Samples were prepared by dropping liposome suspension on carbon coated Cu grid and negatively stained with a 5 % phosphotungstic acid solution. DOX load and release. The ammonium sulfate hydrated liposomes (4.0 mL) were dialyzed in 0.9 % NaCl solution (600 mL) at 4 ℃. The dialysis solution was replaced each hour. The liposomes and DOX (1.0 mL, 2.5 mg/mL) were preheated to 60 ℃ separately. Then the later was dropwise added to the former and incubated at 60 ℃ for 45 min. Subsequently unloaded DOX was removed by Sephadex G50 column. The absorbance of encapsulated DOX Aencapsulated was obtained by using UV-visible spectrophotometer (UV-2450, Shimadzu, Japan) at 480 nm after destructing liposomes by Triton X-100. The encapsulated efficiency EE was calculated as follow (Equation 1): % =

× 100

(1)

Where Atotal was the absorbance of DOX added in solution originally. To study the drug release behavior, two methods were tried, which were release through dialysis tube and without dialysis tube. 1) 1 mL DOX -loaded liposomes with and without UV-irradiation for 10 min were transferred to the dialysis tube (MWCO 14 kDa), and then submerged into 50 mL PB (pH 7.4) solution under stirring at 37 °C. The DOX release from the liposomes was measured by UV-visible spectrophotometer. The release efficiency RE of liposomes was calculated as follow (Equation 2): % =

× 100

(2)

Where was the absorbance at 480nm at time t.


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2) When trapped within liposomes in a high concentration, DOX showed aggregation-caused quenching. When released from liposomes, the fluorescence signal of DOX increased greatly and could be monitored using a spectrofluorimeter (F-4500, Hitachi, Japan). Excitation was fixed at 480 nm and emission spectrum was monitored at 555 nm. After irradiation with 365 nm UV light for 10 min, 20 µL DOX loaded liposomes were added into 980 µL PB (pH 7.4) solution. The emission spectrum was measured over time. The accumulated release efficiency of liposomes was calculated as follow (Equation 3): % =

!"#

× 100

(3)

Where $% was the fluoresence intensity at time t, $& was that of liposomes before irradiation of UV light and $'()*+,-.*%'/ was the data of liposomes treated with Triton X-100. The experiments were conducted in independent triplicate. To measure the effect of irradiation time, liposomes were irradiated with UV light for 30 s or 45 s intermittently. After each irradiation period, 20 µL of sample was removed to 980 µL PB (pH 7.4) solution. The fluorescence intensity was measured and RE could be calculated.

Results and Discussions Photo-responsive properties of Liposome@Gly. The size and zeta potential of liposomes before and after UV-irradiation for 10 min were measured at 25 ℃. Their changes in diameters due to UV-irradiation were shown in Table 1 with their compositions of liposomal membrane. It was found that the liposomes could be controlled to be in uniform size (diameter: 200±25 nm ) after extruding through polycarbonate filters. After UV-irradiation, most liposomes had increases in size, indicating the membrane structure might become looser due to the isomerization of GlyAzoCn. Their zeta potentials were about -33.0±2.0 mV for all liposomes irrelative of UVirradiation. The results suggested that the isomerization of GlyAzoCns had little influence on their zeta potentials, but induced the increase in sizes. Especially, for Lipo7, Lipo8 and Lipo12, their diameters had significant increases, suggesting membrane fusion or aggregation might induced by the looser structure of liposomal bilayer23. Drug encapsulated in these liposomes had been expected to be released quickly from the disturbed liposome bilayers. Figure 1 showed the TEM images and DLS results of Lipo7. Due to UV-irradiation, the single peak of diameter near 200 nm had divided into two peaks at 200 nm and 1000 nm (Figure1b). The average diameter of the Lipo7 increased from about 200 nm to 740 nm. The significant increase in size might indicate not only the expansion but also the fusion or aggregation of liposomes might occur. The size distribution of liposomes verified the vesicle structures existed in both conditions. Figure1a and c gave the morphological changes of liposomes due to UVirradiation. Figure1a showed the spherical shape of liposomes in dark. When irradiated with UV light (Figure1c), the liposomes showed larger sizes and their shapes had somewhat distorted after photoisomerization. In a word, their vesicle structures were still intact after being irradiated by UV light.


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Figure 1. Diameters (b) and TEM images of Lipo7 before (a) and after (c) UV-irradiation

Table 1. Compositions and properties of various liposomes

Liposome

n of GlyAzoCn

Molar ratio

In dark

UV light

GlyAzoCn

HSPC

DSPG

Chol

d /nm

d/nm

Lipo0

0

4

1

5

215.1±1.2

214.7±0.6

Lipo1

0.5

181.2±0.6

178.2±0.4

193.4±1.0

206.0±0.3

1

Lipo2 4

4

1

5

Lipo3

2

211.2±1.0

205.6±0.3

Lipo4

4

198.1±1.2

199.6±0.6

Lipo5

0.5

207.3±1.4

210.2±1.4

216.7±0.6

269.0±4.2

1

Lipo6 7

4

1

5

Lipo7

2

202.1±0.7

734.7±25.1

Lipo8

4

195.2±1.3

827.6±22.3

Lipo9

0.5

200.4±0.5

205.6±0.6

191.2±0.5

202.7±0.5

1

Lipo10 10

4

1

5

Lipo11

2

205.6±0.3

208.2±0.4

Lipo12

4

202.3±0.4

360.8±11.3

The hydrophilicity of GlyAzoCns molecules could be increased upon UV-irradiation because the cis configuration of Azo group tended to be more hydrophilic.24 It was unknown whether GlyAzoCns could be incorporated tightly in liposomal membrane after they changed from trans to cis configuration. Experiments had been done to investigate whether the GlyAzoCns escaped from liposomes. In brief, the liposomes incorporated with various GlyAzoCns ratios were irradiated by UV for 10 min and then dialysed for 2 h. Free GlyAzoCns in the outside solution (dialysate) was judged from the absorption peak at λ≈450 nm. As shown in Figure S15, after UVirradiation, almost all GlyAzoC4 and about half of the GlyAzoC7 escaped from liposomes while the GlyAzoC10 were almost remained in the membrane. This was easy to understand that GlyAzoC10 with the longest carbon chain could maintain the amphipathy to arrange stably in the liposomal bilayer even though the Azo group became hydrophilic. It is important to understand the photoisomerization behavior of the synthesized GlyAzoCns in liposomal bilayer (restricted state) as well as in ethanol (free state).


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Photoisomerization of GlyAzoCns in ethanol and liposome bilayer. Firstly, photoisomerization of free GlyAzoCns were studied when they dissolved in ethanol. Figure2 showed the UV absorption spectra of GlyAzoCns in ethanol at 37 ℃ in dark (black line), upon irradiating with a 365 nm-light (red line) and followed by irradiating with a 470 nm-light (blue line), respectively. The reversible transformation of GlyAzoCns from trans to cis configurations in the ethanol was evident from the decrease of the strong absorption peak at λ≈350 nm (π-π* transition) and the concurrent increase of the weak peak at λ≈450 nm (n-π* transition)25. These absorption spectra clearly illustrated the reversible transformation between cis-isomer and transisomer. The spectra obtained in dark and after UV-Vis light irradiation were found to be almost the same.

a

2.5

GlyAzoC4

b

c

GlyAzoC7

GlyAzoC10

2.0 1.5

In dark UV light Blue light

1.0 0.5 0.0 300

400 500 Wavelength/nm

600

3.0


GlyAzoC4 GlyAzoC7 GlyAzoC10

d

2.5

300

2.0 1.5 1.0 0.5 0.0 0

20

40

60

80

Irradiation time/s

100

120

ln((A0-Aeq)/(At-Aeq))

Absorbance/%

In ethanol

Absorbance/%

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3.0 2.5

600

300


600

e

2.0 1.5 1.0 0.5 0.0 -2 0

2

4

6

8 10 12 14 16 18 20 22

Irradiation time/s

Figure 2. In ethanol at 37℃, the UV/vis absorption spectra of GlyAzoC4 (a), GlyAzoC7 (b) and GlyAzoC10 (c) upon UV-irradiation; the absorbance at λmax (d) and plots of ln((A0-Aeq)/(At-Aeq)) vs. UV-irradiation time t (e) of GlyAzoCns.

The absorbance at the maximum absorption peak (λ=343.5 nm), λmax, was plotted against UV irradiating time (t) and shown in Figure 2d. It took less than 40s for absorbance of GlyAzoC4 to reach a constant value. It was also found, when the length of carbon chain increased, the t value decreased to about 20 s (GlyAzoC10). We assumed the trans-cis transition to be a first order reaction26, 27 as Equation (4).

ln 2 = 34

(4)

2


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Where A0 and Aeq were the absorbance at λmax in ethanol at t=0 and at equilibrium state separately, while At was the absorbance value after irradiation for t. k was the photoisomerization rate and could be obtained from the slope of the line in Figure 2e. The k value of GlyAzoCns in ethanol was listed in Table 2. It was obvious that the k value increased with the carbon chain length of GlyAzoCns.

In bilayer

2.5 2.0

a

GlyAzoC4

b

c

GlyAzoC7

GlyAzoC10

1.5

In dark UV light Blue light

1.0 0.5 0.0 300


600

2.0

300

GlyAzoC4 GlyAzoC7 GlyAzoC10

d 1.5 1.0 0.5 0.0 -50


0

50

100

150

200

Irradiation time/s

250

300

ln((A0-Aeq)/(At-Aeq))

Absorbance/%

Then, the photo-induced isomerization of GlyAzoCns in liposome bilayer was also investigated (Figure 3a,b,c). Similar to those in ethanol, their photoisomerizations occurred reversibly when irradiated with 365 nm-light and 470 nm-light alternately.

Absorbance/%

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600

300


600

2.5 2.0

e

1.5 1.0 0.5 0.0 0

20

40

60

80

100

Irradiation time/s

Figure 3. In bilayer at 37 ℃, the UV/vis absorption spectra of GlyAzoC4(a), GlyAzoC7(b) and GlyAzoC10(c) upon UV-irradiation; the absorbance at λmax (d) and plots of ln((A0-Aeq)/(At-Aeq)) vs. UV-irradiation time t (e) of GlyAzoCns. Molar ratio of HSPC:DSPG:Chol:GlyAzoCn = 4:1:5:2

The values of t for isomerization reaching equilibrium were about 100 s, 120 s and 180 s (Figure 3d), suggesting shorter carbon chain needed more time. And it was easy to found that the necessary time was very long compared with those in ethanol (20 s-40 s). The significant increase in t was thought to be resulted from the interaction between GlyAzoCn and surrounding lipids. When GlyAzoCns incorporated into liposome bilayer, the isomerization process was hindered and slowed. Then, their k values were certainly much smaller than those in ethanol (Table 2). It could be concluded that the liposomal membrane could hinder the isomerization of GlyAzoCns to some extend. However, consistent with that in ethanol, the k value increased with the incremental carbon chain of GlyAzoCns as shown in Figure 3e.


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As shown in Table 2, the molar ratio of GlyAzoCns gave large influence on the photoisomerization process. For GlyAzoC4, a little decrease in photoisomerization speed could be found. The k of GlyAzoC7 decreased to 1/6 of the original and tended to be same values, indicating that the percentage of GlyAzoC7 had no effect on the photoisomerization. I.e., liposomal membrane had the same hindering effect on photoisomerization regardless of the proportion of GlyAzoC7. However, for GlyAzoC10, the isomerization speed decreased gradually with the increase of molar ratio. According to above discussion, GlyAzoC10 did not escape from bilayer after UV-irradiation. Then, the membrane pressure should increase quickly to prevent the isomerization process when there were too many GlyAzoC10 molecules in liposome bilayer. When with a lower content of GlyAzoCns (HSPC:DSPG:Chol:GlyAzoCn=4:1:5:0.5), liposome containing GlyAzoC10 had a larger k value, suggesting its strong resistance against the suppression from the surrounding lipids.

Table 2.The photoisomerization speed of GlyAzoCns in ethanol and liposomal bilayer GlyAzoC4

Mole ratio

GlyAzoC7

-1

GlyAzoC10

k/s

k/s

k/s-1

0.04558

0.11013

0.12323

4:1:5:0.5

0.01433

0.01707

0.05968

4:1:5:1

0.01265

0.01927

0.03970

4:1:5:2

0.01413

0.01917

0.02556

4:1:5:4

0.01011

0.01929

0.01858

In C2H5OH a

-1

a

: HSPC:DSPG:Chol:GlyAzoCn

Photoinduced instantaneous release of Liposome@Gly. The DOX releases of Liposome@Gly in dark and after 10 min UV-irradiation were studied. When the GlyAzoCns were in their trans configurations, they stayed steadily in the liposome bilayer. Upon UVirradiation, their photoinduced isomerizations were expected to provide the perturbation and micro-tunnels in the bilayer to stimulate the leakage of encapsulated ions and small molecules.

With dialysis tube

100 80

b 100

Free DOX Lipo0 Lipo7 in dark Lipo7 UV light

Accumulated release/%

a


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60 40 20 0 0

1

2

Time/h

3

4

Without dialysis tube Lipo7 UV light

80 60 40 20 0 -1

0


1

2

Time/h

3

4

5

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Figure 4. Kinetic release of DOX from liposomes monitored by (a) UV-visible spectrophotometer (with dialysis tube) and (b) spectrofluorimeter (without dialysis tube). Transmembrane pH gradient method was used to load DOX and more than 80 % of DOX could be encapsulated into liposomes. Firstly, the release kinetics of the liposomes was investigated by releasing DOX in a dialysis tube. As shown in Figure 4a, pure liposome (Lipo0) could hold its cargoes for a long time and keep a constant DOX leakage of less than 5 %. Lipo7 had the similar result when released without UV-irradiation, which indicated that the incorporation of GlyAzoC7 had little influence on the stability of liposome in dark. When exposed to UV for 10 min in advance, Lipo7 released about 65 % of encapsulated DOX. Evidently, the release of DOX from Lipo7 could be stimulated by UV-irradiation. From Figure 4a, the release of DOX after UV-irradiation seemed to complete within 2 h. However, since we doubt the DOX release might be hindered by the dialysis tube and the result might not represent its true kinetics, the leakage of free DOX (without liposome) from dialysis tube was also measured. The result was also shown in the Figure 4a, which was found to be similar to that of Lipo7 (with UV-irradiation). The phenomenon suggested that the DOX release might be controlled by the obstruction of dialysis tube and the release of Lipo7 might be much quicker. It was known the DOX encapsulated in liposome was in quenching state and DOX released in solution could be detected directly. So, the later release experiments were recorded by using a fluorophotometer without dialysis tube. As shown in Figure 4b, the fluorescence signal of DOX enhanced immediately after being exposed to UV. And, the fluorescence signal almost unchanged in the next 4 h. These results told us that to study the kinetics of release from these photo-sensitive liposomes was insignificant because the release was an instantaneous behavior. The effect of molecular structure of glycolipids on DOX release. Then, the total release percentages of DOX from the Liposome@Glys were measured immediately after 10 min UVirradiation. As shown in Figure 5, all the Liposome@Glys had very little DOX leakage in dark irrelevant to the proportion of GlyAzoCns, suggesting that these liposomes were almost as stable as the pure liposome (Lipo0). When irradiated with UV, the Liposome@Glys released the DOX abruptly and a maximum release percentage of Lipo8 reached near 70 %. The release percentage was found to increase with the content of GlyAzoCns. Comparing the liposomes incorporated with different glycolipids (Figure 5a, b, c), it was easy to find that the molecular structure of glycolipid induced large difference in photo-triggered release. Among them, insertion of GlyAzoC7 showed to have best effect on controllability of drug release. Figure 5b showed that all the liposomes containing GlyAzoC7 tended to release much more DOX after UV-irradiation than those in dark. It was obvious that after the same UVirradiation time, the more GlyAzoC7 in liposome the more DOX released. It could be speculated that when GlyAzoC7 isomerized, the bulky cis configuration would push the surrounding lipid molecules away and part of them escaped from the bilayer, leaving nano channels on the bilayer surface. The experiments of dialyzing Lipo7 and Lipo8 suspension after being UV-irradiated have verified GlyAzoC7 really escaped from liposomes (data not shown). Furthermore, the DOX could not be released completely, implying the liposome bilayer might restore to its original condition immediately after GlyAzoC7 had escaped. The nano channels on surface of liposomes might be filled by surrounding lipids and then disappeared due to the fluidity of the bilayer. In some sense, the release amount of DOX was decided by the number of GlyAzoC7 in bilayers. However, the


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release of liposomes incorporated with GlyAzoC4 and GlyAzoC10 did not have an obvious improvement upon UV-irradiation. It was thought that their sizes of carbon chain should decide their arranging behavior in liposomal bilayer. Glycolipids with too short carbon chain might make it difficult to push away the lipids at the moment of isomerization but only escape from liposome. For GlyAzoC10, the strong hydrophobicity might keep itself staying in the bilayer after UVirradiation. Both GlyAzoC4 and GlyAzoC10 could not produce nano channel on the bilayer for DOX release. Experiments of dialyzing Liposome@Gly suspension after being UV-irradiated have verified that few of GlyAzoC10 had left from the liposome bilayer. In addition, it could also be considered that the configuration change of GlyAzoCn upon UVirradiation would give disturbance/stimulation to liposomal bilayer and decrease the tightness of the bilayer, which would induce membrane fusion or even aggregation28. As shown in Figure 5, the photo-induced drug releases of Lipo7, Lipo8 and Lipo12 were more efficient, which were found to be consistent with their data of increases in diameter (Table 2), suggesting membrane fusion occurred. In order to prove the membrane fusion of Liposome@Glys, another measurement has been made. Briefly28, DOX-loaded Lipo0 was mixed with DOX-unloaded Lipo7 at the molar ratio of 1:1. The release behaviors of the mixture in dark and after UV-irradiation were investigated (Figure5b). As shown in Figure5b, Lipo0 exposed to UV-light and the mixture in dark could hardly release DOX as expected. However, the mixture reached a 55 % of release ratio immediately after UV-irradiation, which was near the release of Lipo7 shown in Figure5a2. The results indicated that the isomerization of GlyAzoC7 embedded in Lipo7 induced the membrane fusion between Lipo7 and Lipo0 and then stimulated the Lipo0 to release DOX. The fact that the change in property of unilateral membrane also could improve membrane fusion would serve as inspiration to help us understand the function of biomembrane.

80 70 60 50 40 30 20 10 0

a1

a2

GlyAzoC4

Lipo1 Lipo2 Lipo3 Lipo4

b


Total 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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70 60 50 40 30 20 10 0

GlyAzoC7

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Figure 5. a: Total release amount of DOX from Liposome@Glys in dark and after UVirradiation for 10 min; b: Release of DOX from Lipo0 and mixture of Lipo0/Lipo7 (37 ℃).


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Above all, liposome containing GlyAzoC7 had the most improvement in DOX release upon UV-irradiation. GlyAzoC7 was thought to push away the surrounding lipids more effectively and disturb the bilayer resulting in membrane fusion and even liposome aggregation. When incorporated into liposome membrane, a GlyAzoCn with proper carbon length might act effectively as a smart actuator for photo-induced drug release. For incorporation with GlyAzoC10, only Lipo12 (29 % of GlyAzoC10 contained, mol/mol) had an obvious increase in DOX release (Figure 5c). It could be thought, when the proportion of GlyAzoC10 increased enough, even if GlyAzoC10 could not escape from bilayer, the disturbance of isomerization could induce a looser membrane structure and promote DOX release. Actually, the phenomenon of membrane fusion accompanied with isomerization just proved that the membrane structure had become loose28. From above results we could know that the improvement of drug release from Liposome@GlyAzoC7 should be decided by two factors: one was the appearance of nano channel due to the escape of isomerized GlyAzoC7 and another was the membrane fusion resulted from loose membrane structure. However, the DOX release of Liposome@GlyAzoC10 mainly depended on the latter. The effect of cholesterol proportion on DOX release. Due to the fact that cholesterol could adjust the stability of liposomes, the proportion of cholesterol might influence the release behavior of DOX. Lipo7 and Lipo11 were used to study the function of cholesterol. The liposomes mentioned above had a very high cholesterol proportion, i.e., the molar ratio of phospholipids to cholesterol was 1꞉1. In this section, the ratio of phospholipids to cholesterol was changed to 4꞉1 (Lipo7-2 or Lipo11-2) and 10꞉0 (Lipo7-0 or Lipo11-0). Their DOX release experiments were also carried on. As shown in Figure 6, in dark, the reduction of cholesterol seemed to have a little influence on the leakage of DOX, but overall, DOX in these liposomes could be hardly released. However, after UV-irradiation, the DOX release amount increased obviously with the decrease of cholesterol proportion. Especially the release ratio of Lipo7-0 reached almost 100 % immediately. The fluorescence polarization (P) was measured to identify the influence of cholesterol on liposomal fluidity. The P values of Lipo0, Lipo0-2 (phospholipid꞉cholesterol=4꞉1) and Lipo0-0 (phospholipid꞉cholesterol=10꞉0) were 0.413, 0.441 and 0.447 respectively. Since the reciprocal value of polarization (1/P) was defined as the membrane fluidity, it could be known that the liposomal membrane became stable (lower fluidity) when the proportion of cholesterol decreased in our experiment condition. This seems to be counterintuitive according to general knowledge. However, Morgan et al17 found that cholesterol acted as a “buffer” of membrane order. For liquid crystal phase membrane, the addition of cholesterol would help to improve the stability of the liposome, i.e., decreased membrane fluidity. But in gel-phase lipid bilayer, it in turn disordered the rigid phase to increase the fluidity. Here, the liposome composed mainly of HSPC had high phase transformation temperature Tc (about 55 ℃)19 and gel-like phase membrane29. Then the addition of cholesterol increased the membrane fluidity. Results suggested the lipid membrane with a relative lower fluidity seemed to be conducive to promote the drug release. For Lipo7, it was considered that the nano channels on a lower fluidity membrane could be kept for a long time to provide more time to release. From these results it could be concluded that to achieve a complete DOX release from Liposome@Gly, the


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Liposome@Glys should have enough actuators (GlyAzoCn) in their lipid bilayers as well as a lipid membrane with relative low fluidity. As for Lipo11, the release amount also increased with decrease of cholesterol. The GlyAzoC10 could not escape from the bilayer, but the bilayer with low fluidity need more time to restore from looser state. Then, more DOX could release during restoring period.

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Lipo7-2

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Lipo11-2 Lipo11-0

Figure 6. Total release amounts of DOX from liposomes with different proportions of cholesterol. (Irradiation time: 10 min, 37℃)

The effect of irradiation time on DOX release. The isomerization of GlyAzoCns was thought to go on one by one molecule depending on how much optical energy could be obtained. Figure 5b showed that with the same UV-irradiation time, the more GlyAzoC7 in liposome the more DOX released. In this section, release amounts of liposomes were investigated after being irradiated by UV for various periods of time. As shown in Figure 7a, release amount of DOX from Liposome@Gly was found to be controlled by the irradiation time, i.e., increased with UV-irradiation time. For Lipo7 and Lipo8, it was obvious that UV-irradiation for 4 min was enough for photo-induced release. The release amount did not increase after more irradiation only because there was little trans-form Azo group in liposome. Azo group was reported to isomerize reversibly upon Vis and UV-irradiation alternately. But, in this experimental condition, the escape of the GlyAzoC7 from liposomal bilayer made the reversible switch-effect unachievable. However, an intermittent DOX release controlled by UV-irradiation time was expected.


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Figure 7. Accumulated release of DOX from Lipo7 and Lipo8 (a) controlled by the irradiation time, Lipo7 (b) and Lipo7-0 (c) irradiated by UV light for equivalent and intermittent time (37℃).

Further investigation was done by irradiating Liposome@Glys with equivalent and intermittent short time for many times. The DOX release amounts from Lipo7 and Lipo7-0 were measured after being irradiated for 45 s or 30 s every time. As shown in Figure 7b,c, DOX released from liposomes immediately after UV-irradiation every time. Both liposomes showed clear steps of release. The total release percentage from Lipo7 after being irradiated 13 times (total irradiation time: 9.75 min) was consistent with that after successive 10 min of UV-irradiation (See Figure 5). For Lipo7-0, if the sample was irradiated for 45 s every time, it released 65 % of loaded DOX after being irradiated 2 times. Here, the periodic release amounts after being irradiated for 30 s every time were shown. From the results, the DOX was found to be released completely after irradiated 7 times. It could be found that Lipo7-0 only needed 3 min of UV-irradiation to reach the complete release. These results indicated the well reliability and sensitivity of UV-controllable release from Liposome@Glys and verified that the isomerization of GlyAzoCns in liposomal bilayer went on one by one molecule depending on how much optical energy could be obtained. Longer irradiation time meant more cis GlyAzoCns could be isomerized and then more GlyAzoCn molecules could escape from liposomes, which resulted in more membrane fusion and then DOX release amount.


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Meantime, the final accumulated release amounts were certainly decided by the number of GlyAzoCns in liposome. In addition, UV-irradiation had a limitation of penetration and harm to body tissues. How to obtain UV-light in situ might have to consider other methods. The harm of UV-light is not a problem if irradiation time is not very long. Ji et al had used UV light to facilitated DNA release and nuclear entry. They reported that cells were exposed to 365 nm light for 15 min while the UV light had little cytotoxicity to cells.30 In this paper, the short period of UV-irradiation could be thought to be safe. As for the cytotoxicity of GlyAzoCns, papers have reported the azobenzene derivatives had good cell compatibility.31, 32 The systemic toxicity and cell targeting function of Liposome@Glys would be investigated in the future.

Figure 8. The diagram of photoisomerization induced burst release of Liposome@GlyAzoC7.

Conclusions In conclusion, we designed and synthesized a series of novel structured azobenzene derivatives GlyAzoCns (GlyAzoC4, GlyAzoC7 and GlyAzoC10), which had an azobenzene moiety between the galactosyl and various sized carbon chains. Their photo-induced isomerizations showed good reversibility both in ethanol solution and in the liposome bilayer upon irradiated with UV or Vislight alternately. The isomerization process in liposome bilayer was much slower than that in ethanol solution, indicating the hindering effect of surrounding lipids in bilayer. Among all the liposomes, Liposome@GlyAzoC7 presented quite favorable photo-controllable release property. Under UV-irradiation, the GlyAzoCns with a proper sized carbon chain was easy to push away the surrounding lipids and escape from bilayer leaving nano channels inducing loose membrane


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structure, which resulted in membrane fusion and then improvement of DOX release, as depicted in Figure 8. DOX released through the nano channels on Liposome@Gly membrane, which produced by the escape of the GlyAzoCns. The nano channels have disappeared quickly due to the fluidity of the lipid membrane. Higher fluidity resulted in faster membrane recovery and then less DOX release. Since the number of isomerized GlyAzoCn is proportional to the emitting energy of UV, the multimode drug release, such as instantaneous and intermittent release, could be controlled by irradiating time. In a word, to improve DOX release of Liposome@Gly, the number of actuators (GlyAzoCn) in the lipid bilayers as well as the stability of membrane should be increased.

Associated Content Supporting Information Additional synthetic methods(Scheme S1-Scheme S2, Figure S1-Figure S14) and the release of GlyAzoCns from Liposome@Glys upon UV-irradiation (Figure S15). Author information Corresponding Author E-mail: [email protected] (S. Xu)

Acknowledgements This work is supported by the National Natural Science Foundation of China (21276074, 91334203) and the Fundamental Research Funds for the Central Universities. Notes The authors report no conflicts of interest.

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Photocontrollable Intermittent Release of Doxorubicin Hydrochloride

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