Physicochemical Analysis of DPPC and Photopolymerizable

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Physico-Chemical Analysis of DPPC and Photo-Polymerizable Liposomal Binary Mixture for Spatiotemporal Drug Release ahmad kenaan, Cheng Jin, Qi Daizong, Di Chen, Daxiang Cui, and Jie Song Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02144 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 17, 2018

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Analytical Chemistry

Physico-Chemical Analysis of DPPC and Photo-Polymerizable Liposomal Binary Mixture for Spatiotemporal Drug Release Ahmad Kenaan, Cheng Jin, Qi Daizong, Di Chen, Daxiang Cui, Jie Song* *Institute of Nano Biomedicine and Engineering, Shanghai Engineering Research Centre for Intelligent Diagnosis and Treatment Instrument, Department of Instrument Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, 800 Dongchuan RD, Shanghai 200240, P.R. China. ABSTRACT: The development of spatiotemporal drug delivery system with long release profile, high loading efficiency, and robust therapeutic effect is still a challenge. Liposomal nanocarriers have secured a fortified position in the biomedical field over decades. Herein, liposomal binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and photo-polymerizable 1,2bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC) phospholipids were prepared for drug delivery applications. The diacetylenic groups of DC8,9PC produce intermolecular cross-linking following UV irradiation. Exposure of liposomal mixture to 254nm radiation induces a pore within the lipid bilayer, expediting the release of its entrapped 5,6-Carboxyfluorescein dye. The dosage and rate of the released content are highly dependent on the number and size of the induced pore. Photochemical cross-linking studies at different exposure times were reported through the analysis of UV Visible Spectrophotometer, Nano Differential Scanning Calorimetry (NanoDSC), Fourier-Transform Infrared Spectroscopy (FTIR), and Raman Spectroscopy. The optimal irradiation time was established after 8min of exposure inducing lipids cross-linking with minimal oxidative degradation, which plays an essential role in the pathogenesis of numerous diseases due to the formation of primary and secondary oxidation products, accordingly reduces the encapsulated drug therapeutic level.

L

iposomes are simple, concentric bilayered vesicles with a spherical shape enclosing an aqueous compartment. The membranous lipid bilayer composed mainly of cholesterol and natural nontoxic phospholipids.1 Generally, Liposomes particles range in size from tens of nanometers to several micrometers depending on the preparation method.2,3 Liposomes are versatile, non-toxic, biodegradable, hypoallergenic, and Nonimmunogenic.4,5 These unique properties characterize liposomes as an advanced technology in transporting active molecules such as drug to improve its pharmacokinetic effects.6 Thus the development of a spatiotemporal drug delivery system is highly needed to ensure the activity of the drug at the site of action, for the correct time and duration, along with therapeutics improvement. Although several approaches were reported to improve the drug pharmacokinetic effects,7,8 liposomes are still superior. The release mechanism of drug delivery system is classified mainly into ‘’immediate’’ and ‘’modified release’’.9–11 In case of immediate release, the drug is released immediately after administration and it is characterized by immediate action without delaying or prolonging the dissolution or absorption.12,13 In this mechanism, the drug action time is lim-

ited to the time in which its concentration is above the minimal effective concentration (MEC), which is short in case of short biological half-life drugs and thus requires frequent dosing.14 In contrast, modified release has been dedicated to either release the drug after a specific time from the initial administration (Delayed Release), or over a specific interval time at a predetermined rate and concentration (Extended Release) that plays a momentous role in accomplishing a potent therapeutic level.15 In the present work we aim at developing a new liposomal drug delivery system possessing higher encapsulation efficiency, with long release profile over an interval of time competent at satisfying robust therapeutic level. Several stimuli-responsive liposomes systems have been designed to ensure the drug release immediately or with spatiotemporal and dosage control. A variety of physical phenomena were described, including temperature,16–18 magnetic field,19 ultrasound,20 pH,21,22 and light in the ultraviolet,23 visible,32 and near infrared region.25,26 To this end, all these approaches require external stimuli to ensure the release of entrapped molecules from liposomes. However, most of these approaches are limited in their application, since they are not actually controlling the concentration of the active molecules,

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neither maintaining the release profile over a long period of time. Therefore, we highlight herein the possibility of developing a novel drug delivery system in which the drug is encapsulated at high encapsulation efficiency, and released in a predetermined pattern over a specific period of time without using external stimuli. Our new approach is composed of binary mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and photo-polymerizable phospholipid 1,2-bis(10,12tricosadiynoyl)-sn-glycero-3-phosphocholine (DC8,9PC). Phosphocholine lipids (PC) exhibit low electrostatic interaction arises from the zwitterionic nature of the head group.27 Among a variety of phospholipids, DPPC is the strongest surfactant molecules with higher compaction capacity and very low adsorption kinetic.28–30 Pure DPPC liposomes exhibit content leakage at a lower rate and concentration.26,27,31 DPPC/DC8,9PC liposomes were prepared previously for triggered release of calcein.26 Interestingly, DC8,9PC possesses diacetylenic groups in their aliphatic chain that are used to produce intermolecular cross-linking under UV irradiation aimed at achieving greater molecular cohesion and stability. 32–34 Exposing DPPC/ DC8,9PC liposomes to 254 nm wavelength laser light for 0-45min induces phase boundary defects in the lipid bilayer, resulting in content release that is highly dependent on DPPC/ DC8,9PC molar ratio.26 Diacetylene groups of DC8,9PC undergoes 1,4-additions resulting in cross-linking through the formation of the double bond. Generally, lipids occur as unsaturated fatty acids with at least one double bond, or saturated fatty acids in the absence of double bonds. Under UV irradiation, unsaturated lipids can undergo oxidation reaction which is the main phenomenon responsible for lipid degradation, such that the double bonds react with molecular oxygen through a free radical mechanism, resulting in alteration in their physical and chemical

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properties.35 Thus exposing DC8,9PC liposomes to UV radiations could result in its oxidative damaging (Figure 1). The oxidation process is an essential factor in drug delivery systems since the oxidized liposomes reduce its safety due to the formation of oxidative products such as hydroxyperoxides, ketones, aldehydes, and alcohols, causing the pathogenesis of several diseases.36,37 The efficiency of lipid oxidation is highly dependent on the exposure time, along with the degree of unsaturation such that the oxidation risk increases with the number of double bonds.38 Starting from what have mentioned, liposomal binary mixtures of DPPC and DC8,9PC phospholipids with different molar ratios were prepared and crosslinked at a different UV exposure time. The self-release of an encapsulated 5(6)carboxyfluorescein was investigated over 96h before and after crosslinking. The encapsulation efficiency and zeta potential of the mixtures were studied, along with the transition temperature using differential scanning calorimetry (DSC). Moreover, the effect of UV irradiation on the lipid composition of pure DC8,9PC was monitored through the analysis of DSC, UV Visible Spectrophotometer, Fourier-transform infrared spectroscopy (FTIR), and Raman spectroscopy at different exposure times. Our finding revealed that liposomes are prone to oxidative degradation for longer than 8min of UV exposure.

EXPERIMENTAL SECTION Materials. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3phosphocholine (DC8,9PC) were purchased from Avanti Polar Lipids (Shanghai Benro Chem Co.,Ltd). 5(6)Carboxyfluorescein was purchased from Sigma-Aldrich China, Inc. Chemical substances and solvents were used without further purification.

Figure 1. Schematic illustration of DC8,9PC/DPPC liposomal binary mixtures showing the effect of UV exposure on the dye release profile and liposomes chemical structure. At t=8min, monomeric DC8,9PC/DPPC liposome was converted to its polymeric state, whereas at longer exposure, liposomes photodegradation was accomplished resulting in the damaging of chemical bonds.


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Liposomes Preparation. Liposomes were prepared by thin lipid film hydration method followed by extrusion. Briefly DC8,9PC/DPPC lipids of different ratios (0/100, 25/75, 50/50, 75/25, 100/0) were dissolved in a chloroform in a roundbottom flask. The Solvent was evaporated under reduced pressure at 40°C to generate a thin film, followed by drying overnight under nitrogen to remove traces of chloroform. The resulting thin lipid film was then hydrated with PBS 1x buffer containing 1mM of 5(6)-Carboxyfluorescein, vortexed for 1min, and sonicated for 15min using KQ-500DA singlefrequency ultrasonic cleaner at 450C above the lipid transition temperature. Following hydration, small unilamillar liposomes were obtained by extrusion at room temperature through 100 nm polycarbonate membrane 15 times using a mini-extruder (Avanti Polar Lipids- Shanghai Benro Chem Co.,Ltd). Polymerization was achieved using UVP HL-2000 HybriLinker System. Liposomes were placed in the crosslinker chamber and irradiated with 254nm UV lamp at 25 mW/cm2 intensity. The unloaded 5(6)-Carboxyfluorescein were separated from the loaded liposomes through dialysis (dialysis Membrane MD34mm, MW:7000 KD) at room temperature. Liposomes characterization Dynamic Light scattering (DLS) and Zeta-Potential (ζ) Measurement. The average size of the different liposomes mixtures was measured at 25 °C by dynamic light scattering using Brookhaven instruments NanoBrook Omni before and after polymerization. Three cycles of two minutes were performed to obtain the size from light scattering intensity data. The zeta potential was measured using a Brookhaven instruments NanoBrook 90Plus PALS. Transmission Electron Microscope (TEM. The morphology of monomeric and polymeric DC8,9PC/DPPC liposomes with different molar ratios was visualized using Tecnai G2 Spirit Bio-TWIN transmission electron microscope. 50µL of the liposome was added onto a cupper grid and the excessive material was removed using a filtered paper. The Liposomes were stained with 1% aqueous uranyl acetate and dried in air. Differential Scanning Calorimetry (NanoDSC). The phase transition of monomeric and polymeric DC8,9PC/DPPC liposomes with different molar ratios, along with pure DC8,9PC liposomes at different UV exposure times was examined using NanoDSC from TA instruments. The thermal analyses of the different liposomes were recorded with a heating and cooling rate of 0.1 °C/min. A 900µl aliquot of liposomes was equilibrated at 4° C for 12 hours before starting a heating scan. Fourier-transform infrared spectroscopy (FTIR) and Raman spectroscopy. The effect of UV irradiation on the chemical composition of solid state lipids and liposomes was investigated using Nicolet 6700 FT-IR Spectrometer and Senterra R200 L Raman scattering microscope (Bruker Optik). FTIR spectra of solid state lipids were collected with 32 scans in a 900– 3000 cm-1 range with 2cm-1 resolution. Raman spectra of the liposomes were collected with 128 scans in a 900–2400 cm-1 range with 0.5cm-1 resolution with an excitation wavelength of 532nm and 20 mW laser power.

Carboxyfluorescein self-release. The encapsulation efficiency (EE %) of the different liposomal mixtures was determined by measuring the amount of encapsulated 5,6carboxyfluorescein relative to the total initial amount, using

Varian Cary 50 Probe UV Visible Spectrophotometer after removing the free dye. The amount of encapsulated 5,6carboxyfluorescein was calculated after disrupting the liposome using 1% Triton X-100. The leakage profile of the different liposomes was monitored using UV-Visible Spectrometer over 96 hours without external stimulation.

RESULTS AND DISCUSSION Physico-Chemical analysis. Physico-Chemical cross-linking analysis studies the physical and chemical effects arise from the absorption of light by the exposed liposomes, resulting in structure degradation by means of chemical reactions. In order to obtain a better understanding of the effect of UV irradiation on the liposomal lipid bilayer and optimize the exposure time, several analysis were reported including absorption spectroscopy, Raman spectroscopy, FTIR, and NanoDSC. The formation of polymeric DC8,9PC can be scrutinized by naked eye as the polymer is intensely red colored. The absorption spectrum shown in Figure 2A exhibited a strong absorption band around 490nm after 5min of UV exposure. The maximum absorption intensity was accomplished after 8 to 15 min of irradiation. Interestingly, a diminution in the absorption band was reported at longer exposure. This decrease in the intensity and absorption band arises from photodegradation of the polymeric liposomes caused by photo-oxidation. Photodegradation of polymeric DC8,9PC chemical bonds was confirmed by Raman spectroscopy (Figure 2B). Raman spectra are highly dependent on the intensity of the illuminating light, laser frequency, and polymerization degree. C=C and C≡C stretching arises at different wavenumber depending on the color of the polymer whether it is red or blue.39 The Raman intensity of monomeric DC8,9PC (Before polymerization) was negligible compared to the polymeric spectra (after polymerization). Cross-linking of the terminal diacetylene groups was accomplished after UV treatment that produces internal acetylene groups along with alkenes, giving rise to bands at ~2100cm-1 and 1510 cm-1 respectively which are characteristic of red phase polymer. Raman spectra of polymeric DC8,9PC displayed four major peaks at 1068/1212 cm-1, 1510 cm-1, and 2105cm-1, corresponding to C-C, C=C, and C≡C stretches respectively. Raman intensity at 2105cm-1 and 1510cm-1 demonstrated a maximum at 8min of UV exposure, followed by a significant decrease at longer irradiation time. Interestingly, three new different bands were manifested after 1h of UV irradiation at ~1651cm-1, 1725cm-1, and 1782cm-1. The band at 1651cm-1 is a characteristic of trans double bond and conjugated dienes resulting from the isomerization of double bonds that undergo π → π* electron excitation following absorption of UV light.40–42 The other bands at 1725cm-1 and 1782cm-1 could be explicated by the formation of aldehyde produced through a secondary oxidation process.43 Fourier-Transform Infrared Spectroscopy (FTIR) which is known as a complementary technique to Raman spectroscopy was also used to confirm the oxidative degradation of solid state DC8,9PC lipids following UV irradiation. From Figure 2C, the bands at 2923 cm-1 and 2849 cm-1 refer to the C-H stretching region. The drastic increases in the intensity of these bands after UV exposure assigned to the formation of methylene bands. Similarly the band intensity at 1720 cm-1 was enhanced involving carbonyl stretching vibrations of unsatu-



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Figure 2. Studying the effect of UV exposure time on the physico-chemical properties of pure DC8,9PC lipid bilayer using: (A) UV Visible Spectrophotometer, (B) Raman spectroscopy, (C) Fourier-Transform Infrared Spectroscopy, (D) Differential scanning calorimetry.

rated aldehydes resulting from the breakdown of hydroperoxides or lipid endoperoxides.44 However, the band around 1635 cm−1 exhibits a maximum intensity after 15min of irradiation followed by reduction with longer exposure. This band denotes to C=C stretching arising from intramolecular crosslinking, and the decrease was instigated by photodegradation of the double bond. Similarly, the variation in the band intensity around 1402 cm−1 was assigned to the formation of carboxyl groups following oxidation process.45 Moreover, the enhanced intensity at 1470 cm-1 was allocated to the increase in methyl bands. The changes in the broad band at 1280–1200 cm−1 arises from the hydration–dehydration of phosphonic group of the lipids.45,46 Finally, the enhanced intensity at 1093 cm−1 and 970 cm−1 was assigned to O−O bonds of hydroperoxides47 and trans C=C resulting from the isomerization of double bonds following oxidation process.48 The phase transition behavior of pure DC8,9PC liposomes was examined using NanoDSC showing a highly dependence of the main transitions on the exposure time (Figure 2D). At t=0min, DC8,9PC liposomes undergo single sharp transition at 43.85°C. Interestingly, after 5min of exposure, double transitions were stated at 42.7°C and 44.6°C, suggesting the coexistence of polymeric and monomeric DC8,9PC-rich domains that was not completely crosslinked. At this stage, the transition intensity of monomeric DC8,9PC domains at 44.6°C is

higher compared to polymeric domains at 42.7°C. However, the intensity was surpassed after 8min of exposure, indicating the existence of higher portions of polymerized domains. Interestingly, the transition intensity of the polymeric domains was diminished at longer irradiation in accordance with absorption spectroscopy, Raman spectroscopy, and FTIR analysis. Clearly, these variations in the peak intensities arise from photodegradation of the polymeric liposomes and solid state lipids caused by photo-oxidation at longer exposure time (>8min) and consequently alters their physical behavior and chemical composition. Generally, photo-oxidation occurs when triplet oxygen (3O2) is converted to singlet oxygen (1O2) following UV treatment. The singlet oxygen interacts with liposomes through their double bond resulting in hydroperoxide formation which is identified as the primary lipid oxidation products, in addition to aldehydes, ketones, alcohols, and esters as secondary products.49,50 The loss of unsaturated bonds is the main mechanism characterizing oxidation process. The general mechanism of lipid photo-oxidation is described in Figure S1. Indeed, oxidation level is highly dependent on the exposure time that requires controlling such that the highest efficiency of DC8,9PC cross-linking along with the lowest degradation were accomplished after 8min of UV irradiation.


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Carboxylfluorescein self-release. Starting from the oxidation studies, the encapsulation efficiency and release profile of DC8,9PC/DPPC binary mixture with different lipid ratios were piloted before and after crosslinking at 8 min of UV treatment. The initial concentration of 5(6)-Carboxyfluorescein used for encapsulating the different liposomes is 1mM. The morphology of monomeric and polymeric DC8,9PC/DPPC liposomes was examined by transmission electron microscopy along with in the zeta potential, the average hydrodynamic size using DLS, and the phase transition using nano DSC (Figure 3). TEM images revealed a well dispersed, round-shaped liposomes without changes in their morphology, confirming the maintenance of vesicular structure in the binary system following UV treatment (Figure 3A). The diameter of the different mixtures ranges between 76.68nm and 95.01nm before and after UV exposure. The average size of the examined liposomes by DLS ranges between 90-110nm (Figure 3B), in accordance with TEM measurements. A slight decrease in the size was reported after UV cross-linking providing excellent evidence on the vesicular structure retention of monomeric vesicles following polymerization. This phenomenon explains the formation of pores through which the entrapped material can pass. Zeta potential measures the magnitude of electrostatic or charge repulsion/attraction between particles in a liquid sus-

pension.51 Its measurement demonstrates a detailed insight into the causes of dispersion mechanism, along with flocculation or aggregation.52 The fluidity, permeability, and emulsion stability of liposomal bilayer can also be examined.53,54 The magnitude of negative charges on the liposomal surface was reported in Figure 3C. The incorporation of different percentages of monomeric DC8,9PC induces reduction in the zeta potential compared to pure DPPC. However, after UV exposure, a substantial enhancement was observed due to the retention of the vesicular structure of the polymeric DC8,9PC, such that the monomeric lipids were quenched after polymerization, resulting in enhanced stability compared to the monomeric liposomes. The phase transition behavior of single component liposomes (DPPC and DC8,9PC) along with the binary systems (DC8,9PC/DPPC) was investigated before and after crosslinking (Figure 3D,3E). Prior to polymerization, pure DPPC and monomeric DC8,9PC liposomes undergo a single sharp transition at 41.2°C and 43.85°C respectively incompliance with previous measurements (Figure 3D).26,55 The incorporation of different molar ratios of DC8,9PC results in a double transition peaks at a lower melting temperature compared to pure liposomes. DPPC transitions in the binary mixture did not show any shift after increasing the molar ratio of monomeric DC8,9PC (~37°C), whereas DC8,9PC domains demonstrated a

Figure 3. A) Morphology analysis, B) hydrodynamic radius, C) zeta potential, and D) & E) Melting temperature variation of DC8,9PC/DPPC liposomal binary mixtures before and after 8min of UV exposure.



significant shift at higher temperature. Interestingly, UVexposure of the binary mixtures for 8 min results in a reduction in the transition intensity for both DPPC and DC8,9PC domains (Figure 3E). Liposomes were melted over a broader temperature range with a minor shift in the transition for DPPC and DC8,9PC domains after polymerization. Surprisingly, the transition intensity of DPPC domains in the binary mixture was not affected by their molar ratio (32µJ/s), whereas DC8,9PC domains revealed a substantial shift toward higher temperature. Generally, the transition temperature of lipid bilayers varies with several parameters including the nature of the polar head group, the acyl chain length, the degree of saturation of the hydrocarbon chains, and the nature and ionic strength of the suspension medium.56 Herein, several factors elucidate the shift in the melting temperature along with the intensity and the shape of the transition. The incorporation of different molar ratios of DC8,9PC exerted a positive lateral pressure on lipid membrane, resulting in a substantial slash in the phase transition of DPPC.57,58 The diacetylenic groups in DC8,9PC aliphatic chain induces intermolecular cross-linking under UV irradiation, giving rise to enhanced rigidity compared to monomeric DC8,9PC and consequently broadening of the transition peaks.59 From Figure 4A, pure DPPC liposomes demonstrated low encapsulation efficiency of 5(6)-Carboxyfluorescein (20%). The incorporation of different molar ratios of monomeric DC8,9PC did not show a substantial improvement in the encapsulation efficiency. However, a significant enhancement was accomplished after UV irradiation. The maximum efficiency was reported for 50/50 mixture, whereas a drop was demonstrated after increasing the molar ratio of DC8,9PC. Generally, the loading efficiency and release profile are highly dependent on the nature of entrapped materials, along with the chemical and physical properties of liposomal membrane.60 The UV treatment of the different liposomes produces intramolecular crosslinking resulting in higher molecular cohesion, stability, and membrane fluidity compared to the monomeric liposomes. Higher membrane fluidity tends to decrease the transition temperature of liposomes; hence higher encapsulation efficiency is achieved.55 Pure DC8,9PC liposomes show lower encapsulation efficiency compared to the binary mixture following UV treatment due to the coexistence of specific por-

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tions of monomeric DC8,9PC-rich domains that were not polymerized. The polymerization degree is highly dependent on the exposure time, and the total concentration of the liposomes could be an additional factor. If the liposomal concentration is too high, the particle-particle interactions will prevent the UV radiations from exposing the entire surface. To obtain better understanding, additional experiments are required. Figure 4B reports the self-release profile of 5(6)Carboxyfluorescein maintained over 96h for DC8,9PC/DPPC liposomes following UV irradiation. Pure DPPC tended to release only 36% of its content after 24h, however around 90% of the entrapped fluorescein was released from pure DC8,9PC. The incorporation of different molar ratios of DC8,9PC resulted in a significant enhancement in the liposomal release profile. The rate of release is highly dependent on the concentration of incorporated DC8,9PC lipids. Liposomes containing 25%, 50%, and 75% of DC8,9PC released 66%, 82%, 90% of their content respectively after 24h. After 96h, the rate of 5(6)-Carboxyfluorescein release was slow associated with the first 24h. Figure 4C reports the amount of 5(6)Carboxyfluorescein released from monomeric and polymeric DC8,9PC/DPPC liposomes over 96h. Clearly, the amount of dye released from polymerized liposomes is high compared to pure DPPC and monomeric DC8,9PC/DPPC liposomes. As we have mentioned previously, vesicle leakage is highly dependent on the physical and chemical properties of the entrapped materials and liposomes.56,61 For example, variation in permeability of the same entrapped materials was reported for different vesicles possessing different membrane thickness.62 Moreover, fusion of the lipid membrane caused by vesicle trafficking can induce content leakage. Generally, two ideal mechanisms were distinguished to describe the leakage of liposomal content known as “graded” and “all-or-none”. In “graded” leakage, all the vesicles tend to lose part of their content. However, in “all-or-none”, some vesicles release their content completely, whereas others maintained full. All these factors reveal the differences in the leakage profile of DC8,9PC/DPPC prior and after UV cross-linking. Herein for liposomal systems with ~100% release, the content release is controlled by “All” leakage mechanism, whereas for partially released liposomes, our experimental results cannot distinguish between the two different mechanisms. For better under-

Figure 4. A) Encapsulation efficiency of DC8,9PC/DPPC liposomal binary mixtures before and after crosslinking at 8min of UV exposure. B) Rate of 5(6)-Carboxyfluorescein release from polymeric DC8,9PC/DPPC liposomes. C) The amount of 5(6)-Carboxyfluorescein released from monomeric and polymeric DC8,9PC/DPPC liposomes.


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standing and in order to distinguish between the different mechanisms that control our diverse systems, other experiments are required to quantify the amount of 5(6)Carboxyfluorescein released from a single liposome.63 On the other hand, membrane permeability can be enhanced through biological channels or transporters embedded in lipid membranes.61,64,65 In our binary mixture, liposomes permeability was enhanced through the formation of pores inside the lipid bilayer, induced by the retention of DC8,9PC domains following UV irradiation. The size and the number of induced pore are essential factors affecting the release profile of our binary mixture, however complete release is not necessary attainable. For a charged water soluble molecule, energetic cost is required to cross the membrane.66 At some point, the driving force (e.g. the osmotic gradient) for 5(6)Carboxyfluorescein diffusion may be insufficient to fully crossing the membrane. Thus, the incorporation of 25% and 50% of polymeric DC8,9PC results in 71% and 89% of dye release respectively after 96h, however 96% were released at higher molar ratio. Therefore, the size and the number of the pores should be large enough to ensure equilibration of the 5(6)-Carboxyfluorescein with the external medium. Regarding pure DC8,9PC, the comparison with DC8,9PC/DPPC is unreliable since it demonstrated a different binary system composed of monomeric and polymeric DC8,9PC. However, a new drug delivery system can be realized composed of pure DC8,9PC, polymerized at different UV exposure time.

of liposomes after 8min of exposure, resulting in a substantial alteration in their structural composition. FTIR and Raman spectroscopy revealed an increase in the methyl bands, formation of ketones and aldehydes, hydration–dehydration of the lipid phosphonic group, as well as the formation of trans double bonds arises from the isomerization of cis-double bonds. Moreover, the absorption spectrum of pure DC8,9PC liposomes endorsed their degradation process followed by a diminution of absorption peak.

CONCLUSION

Notes

This study illustrated the development of a novel drug delivery system with high loading efficiency and long self-release profile over an interval of time, competent at satisfying robust therapeutic level. The proposed system is made of binary liposomes with DPPC and photopolymerizable DC8,9PC phospholipid bearing diacetylene groups. Intermolecular cross-linking of diacetylene groups is accomplished following UV irradiation at 254nm wavelength, resulting in pore formation within the lipid bilayer. The induced pore tended to enhance the release profile of liposomal content at different rates depending on the molar ration of DC8,9PC in the binary mixture. Upon 8 minutes of UV exposure, a substantial improvement in the loading efficiency along with the electrostatic stability was accomplished for the binary mixture compared to the pure DPPC and DC8,9PC liposomes. Moreover, the morphology of the treated liposomes was studied using TEM, which confirmed the preservation of the vesicular structure. The formation of oxidative products following UV treatment plays an important role in the pathogenesis of several diseases that limits their application as drug nanocarriers, thus the time of UV exposure must be controlled in photopolymerizable liposomal systems. A detailed study on the effect of UV exposure time on physico-chemical characteristics of pure DC8,9PC liposomes and solid state lipids was reported combining UVVisible Spectrophotometer, NanoDSC, FTIR, and Raman spectroscopy. DSC studies indicated that the incorporation of DC8,9PC with different molar ratios induces lipids melting over a broader temperature range compared to pure lipids. The shape and intensity of the transition are highly dependent on liposomes composition and exposure time, demonstrating a substantial enhancement in the rigidity of liposomes. Regarding pure DC8,9PC, our finding monitored oxidative degradation

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. The general mechanism of lipid photo-oxidation described by the classical free radical chain mechanism that is initiated by UV exposure, the photodegradation of unsaturated lipid that involves the formation of ketone as a secondary product, and the effect of UV exposure time on the oxidation level of liposomes.

AUTHOR INFORMATION Corresponding author Phone: +8618217204516. E-mail: [email protected].

Author contributions All authors have given approval to the final version of the manuscript. The authors declare no competing financial interest.

AKNOWLEDGMENT This work is supported by the National Natural Scientific Foundation of China (Grant Nos. 11761141006 and 21605102), and the Project of Thousand Youth Talents from China.

REFERENCES (1)

(2)

(3)

(4)

(5)

(6) (7)

van Meer, G.; Voelker, D. R.; Feigenson, G. W. Membrane Lipids: Where They Are and How They Behave. Nat. Rev. Mol. Cell Biol. 2008, 9 (2), 112–124. Akbarzadeh, A.; Rezaei-Sadabady, R.; Davaran, S.; Joo, S. W.; Zarghami, N.; Hanifehpour, Y.; Samiei, M.; Kouhi, M.; Nejati-Koshki, K. Liposome: Classification, Preparation, and Applications. Nanoscale Res. Lett. 2013, 8 (1), 102. Hupfeld, S.; Holsæter, A. M.; Skar, M.; Frantzen, C. B.; Brandl, M. Liposome Size Analysis by Dynamic/Static Light Scattering upon Size Exclusion-/Field FlowFractionation. J. Nanosci. Nanotechnol. 2006, 6 (9), 3025–3031. Grijalvo, S.; Mayr, J.; Eritja, R.; Díaz, D. D. Biodegradable Liposome-Encapsulated Hydrogels for Biomedical Applications: A Marriage of Convenience. Biomater. Sci. 2016, 4 (4), 555–574. Aqil, F.; Munagala, R.; Jeyabalan, J.; Vadhanam, M. V. Bioavailability of Phytochemicals and Its Enhancement by Drug Delivery Systems. Cancer Lett. 2013, 334 (1), 133–141. Langner, M.; Kral, T. E. Liposome-Based Drug Delivery Systems. Pol. J. Pharmacol. 51 (3), 211–222. Rubert, M.; Dehli, J.; Li, Y.-F.; Taskin, M. B.; Xu, R.; Besenbacher, F.; Chen, M. Electrospun PCL/PEO



(8)

(9)

(10)

(11)

(12)

(13)

(14)

(15)

(16)

(17)

(18)

(19)

(20)

(21)

(22)

(23)

Coaxial Fibers for Basic Fibroblast Growth Factor Delivery. J. Mater. Chem. B 2014, 2 (48), 8538–8546. Taskin, M. B.; Xu, R.; Zhao, H.; Wang, X.; Dong, M.; Besenbacher, F.; Chen, M. Poly(norepinephrine) as a Functional Bio-Interface for Neuronal Differentiation on Electrospun Fibers. Phys. Chem. Chem. Phys. 2015, 17 (14), 9446–9453. Löbenberg, R.; Kim, J. S.; Amidon, G. L. Pharmacokinetics of an Immediate Release, a Controlled Release and a Two Pulse Dosage Form in Dogs. Eur. J. Pharm. Biopharm. 2005, 60 (1), 17–23. Leucuta, S. E. Drug Delivery Systems with Modified Release for Systemic and Biophase Bioavailability. Curr. Clin. Pharmacol. 2012, 7 (4), 282–317. Wen, H.; Jung, H.; Li, X. Drug Delivery Approaches in Addressing Clinical Pharmacology-Related Issues: Opportunities and Challenges. AAPS J. 2015, 17 (6), 1327–1340. Schreiner, T.; Schaefer, U. F.; Loth, H. Immediate Drug Release from Solid Oral Dosage Forms. J. Pharm. Sci. 2005, 94 (1), 120–133. Nyol, S.; Gupta, M. Immediate Drug Release Dosage Form: A Review. J. Drug Deliv. Ther. 2013, 3 (2), 155– 161. Perrie, Y.; Rades, T. FASTtrack Pharmaceutics: Drug Delivery and Targeting, illustrate.; Pharmaceutical Press: london, 2012. Andrade, C. Sustained-Release, Extended-Release, and Other Time-Release Formulations in Neuropsychiatry. J. Clin. Psychiatry 2015, e995–e999. Chen, K.-J.; Liang, H.-F.; Chen, H.-L.; Wang, Y.; Cheng, P.-Y.; Liu, H.-L.; Xia, Y.; Sung, H.-W. A Thermoresponsive Bubble-Generating Liposomal System for Triggering Localized Extracellular Drug Delivery. ACS Nano 2013, 7 (1), 438–446. Al-Ahmady, Z. S.; Al-Jamal, W. T.; Bossche, J. V.; Bui, T. T.; Drake, A. F.; Mason, A. J.; Kostarelos, K. Lipid– Peptide Vesicle Nanoscale Hybrids for Triggered Drug Release by Mild Hyperthermia in Vitro and in Vivo. ACS Nano 2012, 6 (10), 9335–9346. Needham, D.; Dewhirst, M. W. The Development and Testing of a New Temperature-Sensitive Drug Delivery System for the Treatment of Solid Tumors. Adv. Drug Deliv. Rev. 2001, 53 (3), 285–305. Plassat, V.; Wilhelm, C.; Marsaud, V.; Ménager, C.; Gazeau, F.; Renoir, J.-M.; Lesieur, S. Anti-EstrogenLoaded Superparamagnetic Liposomes for Intracellular Magnetic Targeting and Treatment of Breast Cancer Tumors. Adv. Funct. Mater. 2011, 21 (1), 83–92. Huang, S.-L.; MacDonald, R. C. Acoustically Active Liposomes for Drug Encapsulation and UltrasoundTriggered Release. Biochim. Biophys. Acta - Biomembr. 2004, 1665 (1–2), 134–141. Sánchez, M.; Aranda, F. J.; Teruel, J. A.; Ortiz, A. New pH-Sensitive Liposomes Containing Phosphatidylethanolamine and a Bacterial Dirhamnolipid. Chem. Phys. Lipids 2011, 164 (1), 16–23. Ju, L.; Cailin, F.; Wenlan, W.; Pinghua, Y.; Jiayu, G.; Junbo, L. Preparation and Properties Evaluation of a Novel pH-Sensitive Liposomes Based on ImidazoleModified Cholesterol Derivatives. Int. J. Pharm. 2017, 518 (1–2), 213–219. Mueller, A.; Bondurant, B.; O’Brien, D. F. VisibleLight-Stimulated Destabilization of PEG-Liposomes.

(24)

(25)

(26)

(27)

(28) (29)

(30)

(31)

(32)

(33)

(34)

(35)

(36)

(37)

(38) (39)

Page 8 of 10 Macromolecules 2000, 33 (13), 4799–4804. Anderson, V. C.; Thompson, D. H. Triggered Release of Hydrophilic Agents from Plasmalogen Liposomes Using Visible Light or Acid. Biochim. Biophys. Acta 1992, 1109 (1), 33–42. Chuang, E.-Y.; Lin, C.-C.; Chen, K.-J.; Wan, D.-H.; Lin, K.-J.; Ho, Y.-C.; Lin, P.-Y.; Sung, H.-W. A FRETGuided, NIR-Responsive Bubble-Generating Liposomal System for in Vivo Targeted Therapy with Spatially and Temporally Precise Controlled Release. Biomaterials 2016, 93, 48–59. Yavlovich, A.; Singh, A.; Tarasov, S.; Capala, J.; Blumenthal, R.; Puri, A. Design of Liposomes Containing Photopolymerizable Phospholipids for Triggered Release of Contents. J. Therm. Anal. Calorim. 2009, 98 (1), 97–104. Wang, F.; Liu, J. Self-Healable and Reversible Liposome Leakage by Citrate-Capped Gold Nanoparticles: Probing the Initial Adsorption/desorption Induced Lipid Phase Transition. Nanoscale 2015, 7 (38), 15599–15604. Singh, S. Human Respiratory Viral Infections, 1st ed.; CRC Press: Boca Raton, 2014. Nguyen, P. N.; Waton, G.; Vandamme, T.; Krafft, M. P. Reversing the Course of the Competitive Adsorption between a Phospholipid and Albumin at an Air–water Interface. Soft Matter 2013, 9 (42), 9972. Albon, N.; Sturtevant, J. M. Nature of the Gel to Liquid Crystal Transition of Synthetic Phosphatidylcholines. Proc. Natl. Acad. Sci. 1978, 75 (5), 2258–2260. Li, J.; Wang, X.; Zhang, T.; Wang, C.; Huang, Z.; Luo, X.; Deng, Y. A Review on Phospholipids and Their Main Applications in Drug Delivery Systems. Asian J. Pharm. Sci. 2015, 10 (2), 81–98. Puri, A.; Yavlovich; Viard; Gupta; Sine; Vu; Blumenthal; Tata, D. Low-Visibility Light-Intensity Laser-Triggered Release of Entrapped Calcein from 1,2Bis (Tricosa-10,12-Diynoyl)-Sn-Glycero-3Phosphocholine Liposomes Is Mediated through a Type I Photoactivation Pathway. Int. J. Nanomedicine 2013, 2575. Kenaan, A.; Nguyen, T. D.; Dallaporta, H.; Raimundo, J.-M.; Charrier, A. M. Subpicomolar Iron Sensing Platform Based on Functional Lipid Monolayer Microarrays. Anal. Chem. 2016, 88 (7), 3804–3809. El Zein, R.; Dallaporta, H.; Charrier, A. M. Supported Lipid Monolayer with Improved Nanomechanical Stability: Effect of Polymerization. J. Phys. Chem. B 2012, 116 (24), 7190–7195. Okada, S.; Peng, S.; Spevak, W.; Charych, D. Color and Chromism of Polydiacetylene Vesicles. Acc. Chem. Res. 1998, 31 (5), 229–239. Lee, R. F. Photo-Oxidation and Photo-Toxicity of Crude and Refined Oils. Spill Sci. Technol. Bull. 2003, 8 (2), 157–162. Olpin, S. E. Fatty Acid Oxidation Defects as a Cause of Neuromyopathic Disease in Infants and Adults. Clin. Lab. 2005, 51 (5–6), 289–306. Feiner, G. Meat and Fat. In Salami; Elsevier, 2016; pp 3– 30. Itoh, K.; Nishizawa, T.; Yamagata, J.; Fujii, M.; Osaka, N.; Kudryashov, I. Raman Microspectroscopic Study on Polymerization and Degradation Processes of a Diacetylene Derivative at Surface Enhanced Raman Scattering Active Substrates. 1. Reaction Kinetics. J.


Page 9 of 10 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

(40) (41)

(42)

(43)

(44)

(45)

(46)

(47)

(48)

(49)

(50)

(51)

(52)

(53)

(54) (55)

(56)

Analytical Chemistry Phys. Chem. B 2005, 109 (1), 264–270. Frankel, E. N. Hydroperoxide Formation. In Lipid Oxidation; Elsevier, 2012; pp 25–50. Sadeghi-Jorabchi, H.; Hendra, P. J.; Wilson, R. H.; Belton, P. S. Determination of the Total Unsaturation in Oils and Margarines by Fourier Transform Raman Spectroscopy. J. Am. Oil Chem. Soc. 1990, 67 (8), 483– 486. Agbenyega, J. K.; Claybourn, M.; Ellis, G. A Study of the Autoxidation of Some Unsaturated Fatty Acid Methyl Esters Using Fourier Transform Raman Spectroscopy. Spectrochim. Acta Part A Mol. Spectrosc. 1991, 47 (9– 10), 1375–1388. Mirghani, M. E. S.; Man, Y. B. C.; Jinap, S.; Baharin, B. S.; Bakar, J. Rapid Method for Determining Malondialdehyde as Secondary Oxidation Product in Palm Olein System by Fourier Transform Infrared Spectroscopy. Phytochem. Anal. 2002, 13 (4), 195–201. Kiwi, J.; Nadtochenko, V. New Evidence for TiO 2 Photocatalysis during Bilayer Lipid Peroxidation. J. Phys. Chem. B 2004, 108 (45), 17675–17684. Nadtochenko, V. A.; Rincon, A. G.; Stanca, S. E.; Kiwi, J. Dynamics of E. Coli Membrane Cell Peroxidation during TiO2 Photocatalysis Studied by ATR-FTIR Spectroscopy and AFM Microscopy. J. Photochem. Photobiol. A Chem. 2005, 169 (2), 131–137. Kinder, R.; Ziegler, C.; Wessels, J. M. GammaIrradiation and UV-C Light-Induced Lipid Peroxidation: A Fourier Transform-Infrared Absorption Spectroscopic Study. Int. J. Radiat. Biol. 1997, 71 (5), 561–571. Merle, C.; Laugel, C.; Baillet-Guffroy, A. Effect of UVA or UVB Irradiation on Cutaneous Lipids in Films or in Solution. Photochem. Photobiol. 2010, 86 (3), 553–562. Le Dréau, Y.; Dupuy, N.; Gaydou, V.; Joachim, J.; Kister, J. Study of Jojoba Oil Aging by FTIR. Anal. Chim. Acta 2009, 642 (1–2), 163–170. Choe, E.; Min, D. B. Mechanisms and Factors for Edible Oil Oxidation. Compr. Rev. Food Sci. Food Saf. 2006, 5 (4), 169–186. Ghnimi, S.; Budilarto, E.; Kamal-Eldin, A. The New Paradigm for Lipid Oxidation and Insights to Microencapsulation of Omega-3 Fatty Acids. Compr. Rev. Food Sci. Food Saf. 2017, 16 (6), 1206–1218. Sou, K.; Tsuchida, E. Electrostatic Interactions and Complement Activation on the Surface of Phospholipid Vesicle Containing Acidic Lipids: Effect of the Structure of Acidic Groups. Biochim. Biophys. Acta - Biomembr. 2008, 1778 (4), 1035–1041. Larsson, M.; Hill, A.; Duffy, J. Suspension Stability; Why Particle Size, Zeta Potential, and Rheology Are Important. Ann Trans Nord. Rheol Soc 2012, 20, 209– 2014. Kirby, C.; Clarke, J.; Gregoriadis, G. Effect of the Cholesterol Content of Small Unilamellar Liposomes on Their Stability in Vivo and in Vitro. Biochem. J. 1980, 186 (2), 591–598. Lasic, D. D. Liposomes: From Physics to Applications; Elsevier: Amsterdam, 1993. Pantusa, M.; Bartucci, R.; Marsh, D.; Sportelli, L. Shifts in Chain-Melting Transition Temperature of Liposomal Membranes by Polymer-Grafted Lipids. Biochim. Biophys. Acta - Biomembr. 2003, 1614 (2), 165–170. Mozafari, M. R. Nanoliposomes: Preparation and Analysis; Volkmar Weissig, Ed.; Humana Press, 2010.

(57)

(58) (59)

(60)

(61)

(62)

(63)

(64)

(65)

(66)

Montesano, G.; Bartucci, R.; Belsito, S.; Marsh, D.; Sportelli, L. Lipid Membrane Expansion and Micelle Formation by Polymer-Grafted Lipids: Scaling with Polymer Length Studied by Spin-Label Electron Spin Resonance. Biophys. J. 2001, 80 (3), 1372–1383. Marsh, D. Elastic Constants of Polymer-Grafted Lipid Membranes. Biophys. J. 2001, 81 (4), 2154–2162. Yager, P.; Schoen, P. E.; Davies, C.; Price, R.; Singh, A. Structure of Lipid Tubules Formed from a Polymerizable Lecithin. Biophys. J. 1985, 48 (6), 899–906. Stamp, D.; Juliano, R. L. Factors Affecting the Encapsulation of Drugs within Liposomes. Can. J. Physiol. Pharmacol. 1979, 57 (5), 535–539. Ladokhin, A. S.; Wimley, W. C.; White, S. H. Leakage of Membrane Vesicle Contents: Determination of Mechanism Using Fluorescence Requenching. Biophys. J. 1995, 69 (5), 1964–1971. Paxton, W. F.; Price, D.; Richardson, N. J. Hydroxide Ion Flux and pH-Gradient Driven Ester Hydrolysis in Polymer Vesicle Reactors. Soft Matter 2013, 9 (47), 11295. Sun, B.; Chiu, D. T. Determination of the Encapsulation Efficiency of Individual Vesicles Using Single-Vesicle Photolysis and Confocal Single-Molecule Detection. Anal. Chem. 2005, 77 (9), 2770–2776. Mulkidjanian, A. Y.; Galperin, M. Y.; Koonin, E. V. CoEvolution of Primordial Membranes and Membrane Proteins. Trends Biochem. Sci. 2009, 34 (4), 206–215. Apellániz, B.; Nieva, J. L.; Schwille, P.; García-Sáez, A. J. All-or-None versus Graded: Single-Vesicle Analysis Reveals Lipid Composition Effects on Membrane Permeabilization. Biophys. J. 2010, 99 (11), 3619–3628. Boron, W. F.; Boulpaep, E. L. Medical Physiology : A Cellular and Molecular Approach; Saunders Elsevier: Philadelphia, 2016.



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Physicochemical Analysis of DPPC and Photopolymerizable

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