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Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug

Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug Liposome is a promising carrier system for delivering bioactive molecules. However, the successful delivery of pH-sensitive molecules is still limited by the intrinsic instability of payloads in physiological environment. Herein, we developed a special liposome system that possesses an acidic micro-environment in the internal aqueous chamber to improve the chemical stability of pH-sensitive payloads. Curcumin-loaded liposomes (Cur-LPs) with varied internal pH values (pH 2.5, 5.0, or 7.4) were prepared. These Cur-LPs have similar particle size of 300 nm, comparable physical stabilities and analogous in vitro release profiles. Interestingly, the chemical stability of liposomal curcumin in 50% fetal bovine serum and its anticancer efficacy in vitro are both micro-environmental pH-dependent (Cur-LP-2.5 > Cur-LP-5.0 > Cur-LP-7.4). This serum stability still has space to be further enhanced to improve the applicability of Cur-LP. In conclusion, creating an acidic micro-environment in the internal chamber of liposome is feasible and efficient to improve the chemical stability of pH-sensitive payloads. Keywords: Liposomes, Nanoparticles, Drug delivery, Controlled release, Curcumin Background values). Generally, liposome is prepared in a neutral buffer Liposome, an artificial membrane vehicle, has shown solution and thus the loaded drug molecules are also in a great potentials in drug delivery due to its drug loading neutral environment after incorporation into liposome. capacity, biodegradability, and biocompatibility [1–4]. Accordingly, those molecules which are only stable in The classic liposome is similar with living cells in struc- acidic environment would be still instable even in the ture, typically consisting of a phospholipid bilayer and form of liposome. Therefore, development of a novel an aqueous inner chamber [5–7]. Due to this structure, approach for enhancing the stability of pH-sensitive drugs liposome is able to solubilize the insoluble drug molecules is of great importance for successful delivery of these and prevent the loaded drug from the harsh physiological payloads by liposome. environment [8–10]. In addition, the surface of liposome As mentioned above, liposome has an aqueous space can be modified to prolong the blood circulation time in its inner chamber, which can be used to provide drug and/or target specific tissues [11–15]. With these above- payloads with an acidic micro-environment (Fig. 1). In mentioned advantages, various liposome systems have this present work, we use curcumin as a model drug and been clinically approved [8, 9, 16]. aim to provide a novel approach for enhancing the Although the delivery of many drugs has been improved chemical stability of drug molecules loaded in liposome. by incorporation into liposome, the delivery of some It is well known that curcumin is a lipophilic molecule pH-sensitive drugs is still limited by the instability of drug and has been extensively used in food, medicines, and molecule itself in physiological environment (neutral pH cosmetics due to its various bioactivities [17–21]. How- ever, its delivery is highly limited by its insolubility and * Correspondence: dentistcai@hotmail.com; qiangpengzz@scu.edu.cn; instability in biological fluids [22–25]. So far, it is yet to lijm2002@163.com fulfill its clinical promise in part due to pH-mediated Equal contributors instability [26]. Therefore, curcumin is a suitable model State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Block 3, Renmin Road South, Chengdu 610041, drug for this work. China © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 2 of 8 Fig. 1 Schematics of the liposome with varied micro-environmental acidity in its inner aqueous chamber Methods The size and zeta potential of LP were determined by Materials dynamic light scattering (DLS) and electrophoretic light Phospholipids (soybean lecithin for injection use) were scattering (ELS), respectively, using ZetasizerNano ZS90 purchased from Shanghai Tai-Wei Pharmaceutical Co., (Malvern Instruments Ltd., Malvern, UK) at 25 °C [29]. Ltd., (Shanghai, China). Cholesterol was obtained from The measurement cycle was automatically determined by Amresco (Solon, OH, USA). Poloxamer 188 (F68) was the instrument system. The particle size was presented by kindly donated by BASF (China) Co., Ltd., (Shanghai, intensity distribution, and the size distribution was evalu- China). Curcumin was supplied by Sigma (St. Louis, MO, ated by polydispersity index (PDI). USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). All other chemical reagents Encapsulation Efficiency (EE) Determination used in this study were of analytical grade or better. EE, an important parameter for quality control, is of great significance in developing liposome-based delivery Preparation of Curcumin-Loaded Liposomes (Cur-LPs) systems. The EE determination was based on the high The liposomes with varied micro-environmental pH values speed centrifugation method. Briefly, 100 μl Cur-LPs were prepared using evaporation method according to pre- was centrifuged at the low speed (3000 rpm, 5 min) to vious works with some modifications [27, 28]. Briefly, precipitate non-dissolved free curcumin, and 50 μl phospholipids (75 mg) and cholesterol (5 mg) were dis- supernatant was subjected to high-speed centrifugation solved in 0.5 ml ethanol containing 2 mg/ml curcumin. (16 krpm, 10 min) to separate Cur-LPs from the tiny The ethanol solution was mixed with 5 ml 0.001 M PBS dissolved curcumin. The pellets were re-suspended in containing 1% (w/v) F68 that served as a surfactant to 500 μl PBS (i.e., 10-fold dilution), an aliquot of 10 μlof narrow the size distribution. After magnetically stirring for which was mixed with 300 μl ethanol by vortex and son- 1 min (constant temperature magnetic mixer, DF-101S, ication for 30 s. The fluorescent intensity of curcumin in Zhengzhou Greatwall Scientific Industrial and Trade Co., the resultant solution was determined (excitation wave- Ltd., Zhengzhou, China), the resultant emulsion was evap- length (Ex), 458 nm; emission wavelength (Em), 548 nm) orated under vacuum and dark for 30 min at 35 °C to and presented as F , i.e., the fluorescent intensity of remove ethanol. The acidity in the inner chamber of Cur- encapsulated curcumin. Another 50 μl of fresh Cur-LP LP was adjusted via using PBS with varied pH values of containing encapsulated and free curcumin was also 2.5, 5.0, or 7.4 during preparation. The resultant suspen- diluted by 10-fold with PBS, and 10 μl of the diluted sion was centrifuged at a low speed (3000 rpm, 5 min) to solution were mixed with 300 μl ethanol. The fluores- precipitate free curcumin. The supernatant was then cen- cent intensity of the resultant solution was measured trifuged at a high speed (16 krpm, 10 min), and the pellets and presented as F , i.e., the fluorescent intensity of total were re-suspended in PBS (pH 7.4) before further use. curcumin. The EE was therefore calculated with the This procedure provided these LPs with an identical exter- following equation: EE = F /F . e t nal environment. The obtained liposomes with different micro-environmental pH values were presented as Cur- Scanning Electron Microscopy (SEM) LP-2.5, Cur-LP-5.0, and Cur-LP-7.4, respectively. Blank The morphology of LP was observed by the scanning elec- liposomes were also fabricated as above. tron microscopy (SEM, INSPECT F, FEI, Netherlands) [30]. Briefly, the LP suspension was 100-fold diluted with Characterization of Liposome distilled water, and one drop of the diluted suspension was The hydrodynamic size, size distribution, and zeta poten- placed on a clean glass sheet. After air-drying, the sample tial are the three basic parameters for liposome systems. was coated with gold right before SEM. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 3 of 8 Physical Stability of Liposomes onto the 96-well cell culture plates at a density of 10,000 Physical stability is a very meaningful parameter for stor- cells per well and cultured under standard conditions age and transportation of a colloidal system. The phys- (37 °C/5% CO ) for 24 h in PRIM-1640 culture medium ical stability of liposome was presented by colloidal supplemented with 10% FBS. Subsequently, the culture stability and investigated according to a previous method medium was removed and cells were washed with PBS. [31]. Briefly, 100 μl of LP was added to tubes and kept at The Cur-LPs were diluted in serum-free culture medium 37 °C. At different time intervals, the LP size was mea- (4 μg/ml curcumin) and added to cells, followed by sured and compared to the initial size so as to indicate continuous incubation for 1 and 3 days at 37 °C. The the thermodynamic stability. In addition, another 300 μl OD value of viable cells was measured by cck-8 assay. of LP were also added to tubes and kept at 37 °C. At the The cells treated with blank culture medium served as same time intervals, 100 μl of the upper layer liquid control and cell viability (%) was the OD value percent- were collected. The transmittance of the collected speci- age of specimens relative to control. mens was measured at 550 nm and compared to the initial value so as to indicate the kinetic stability. Statistics All the data are presented as mean ± sd (standard devi- In Vitro Release ations). The differences between two groups, analyzed The release profile of liposome plays an important role by the Student’s t test, were considered to be statisti- in predicting in vivo fate and efficacy of liposome. The cally significant when the p value was less than 0.05. in vitro release of curcumin from Cur-LP was studied using the dynamic dialysis method [32]. Briefly, 1 ml of Results and Discussion each Cur-LP was added into a dialysis bag (molecular Characterization of Liposome weight cutoff, 10 kD), which was used to retain liposome The micro-environmental pH of liposome refers to but keep the released curcumin molecules permeable. theacidity in theinner aqueouschamber of liposome The specimen-loaded dialysis bag was soaked in 4 ml (Fig. 1), which is different from the pH in the exter- release medium (0.001 M PBS containing 0.1% Tween nal environment. In this work, the external environ- 80, pH 7.4), and the release study was conducted away mental pH of all liposome suspensions was 7.4 unless from light (37 °C, 100 rpm). At each fixed time interval, otherwise stated. the release medium was collected and replaced with Particle size, zeta potential, and encapsulation effi- 4 ml fresh medium so as to simulate sink conditions. ciency (EE) are important parameters for quality con- The collected medium was diluted to 5 ml with PBS and trol of liposome. The size of three Cur-LPs was similar further diluted by 15-fold with ethanol. The curcumin in to each other (around 300 nm, Fig. 2a). The PDI of the resultant solution was quantified by fluorescence each formulation was lower than 0.2, indicating a nar- spectrophotometry (Ex 458 nm, Em 548 nm). In row size distribution. Interestingly, the negative zeta addition, curcumin powder was dissolved in the above potential of Cur-LP-7.4 (−9 mV) is significantly lower release medium, and the release of curcumin solution than that of the other two Cur-LPs (~−18 mV). Usually, was conducted at pH 7.4 to investigate whether the the negative zeta potential would decrease and even dialysis bag would retain curcumin molecules. convert to positive value with decreasing the pH of disperse phase due to the increase of H concentration. Chemical Stability of Liposomal Curcumin We indeed observed this phenomenon when preparing Chemical stability is a key parameter for predicting drug Cur-LPs in non-buffer HCl/NaOH solutions with pH of metabolism, efficacy, and toxicity. The chemical stability 2.5, 5.0, and 7.4 (Fig. 2b). In the case of PBS, however, 3− 2− − of Cur-LPs was examined in 50% FBS. Briefly, 100 μlof the existence of PO ,HPO ,and/or H PO and their 4 4 2 4 Cur-LPs were diluted by 10-fold with PBS (pH 7.4) and interaction with LP may lead to more complicated situ- then mixed with 1 ml FBS. The specimens were shaken on ations and different results. It is well recognized that a horizontal shaker away from light (37 °C, 100 rpm). At zeta potential plays key roles in maintaining the fixed time intervals, an aliquot of 10 μl of specimen was colloidal stability of nano-scaled suspension. In general, collected and mixed with 300 μl ethanol immediately a higher absolute value of zeta potential leads to a more followed by centrifugation (16 krpm, 5 min). The remained stable colloidal suspension system. curcumin in the supernatant was quantified as above. EE is of concern during liposome development. Usually, increasing EE is important for reducing cost and enhan- In Vitro Anticancer Efficacy cing efficacy. In this work, the EE of Cur-LP-2.5 is 74% The preliminary anticancer efficacy of the three Cur-LPs (Fig. 2c), which is the highest among Cur-LP-5.0 (45%) was investigated using human liver hepatocellular and Cur-LP-7.4 (64%), indicating that Cur-LP-2.5 is the carcinoma HepG2 cells. Briefly, HepG2 cells were plated best formulation for delivering curcumin from the point Shao et al. Nanoscale Research Letters (2017) 12:504 Page 4 of 8 particle distribution. The LP-7.4 also shows a sphere-like shape, but adhesion among particles can be clearly observed (Fig. 3c), indicating that the drying process during SEM specimen preparation would lead to aggre- gation of LP-7.4. This may be due to the relatively low absolute value of zeta potential of LP-7.4 (Fig. 2a). Add- itionally, the particle size measured by SEM is smaller than the hydrodynamic size measured by DLS, which is due to the loss of hydration shell of liposome after drying process for SEM. Physical Stability of Liposome Liposome is a colloidal system and its physical stability can be presented by colloidal stability, which has sub- stantial impacts on liposome storage and further applica- tions [34, 35]. Particle aggregation (thermodynamic instability) and sedimentation (kinetic instability) are the two essential aspects of colloidal instability. Aggregation leads to a larger apparent size and sedimentation leads to changes in transmittance of suspension. More import- antly, size increase can directly affect the efficacy of nano-systems since particle size has been shown to have great impacts on cellular uptake, cytotoxicity, pharmaco- kinetic profile, and tissue distribution [36, 37]. Here, we examined the aggregation and sedimenta- tion properties of three liposome systems to indicate their thermodynamic and kinetic stability, respectively. As shown in Fig. 4a, the three LPs showed no substan- tial changes in hydrodynamic size within 72 h, indicat- ing that all these LPs have a very high thermodynamic stability. Meanwhile, the transmittance change of all the three LPs was less than 10% (Fig. 4b), indicating little particle sedimentation and thus a high kinetic stability. These results suggestthatthe threeLPs have an excellent colloidal stability within 72 h, and the micro-environmental pH has no influence on physical stability of liposome. In Vitro Release Drug release profile from liposome is usually examined to evaluate formulation quality, provide reference for Fig. 2 Physicochemical characterization of liposomes. a Hydrodynamic size and zeta potential of Cur-LPs fabricated in PBS with pH 2.5, 5.0, dosage regimen, and predict the effectiveness in vivo. In and 7.4, respectively. b Zeta potential of Cur-LPs fabricated in HCl/ general, almost all liposomal systems have a sustained NaOH solutions with pH 2.5, 5.0, and 7.4, respectively. c Encapsulation drug release property. Here, we examined the in vitro efficiency of Cur-LPs prepared in PBS. Data presented as mean ± sd release behavior of three Cur-LPs in PBS (pH 7.4). (n = 3). Statistical significance between groups: ***p <0.001 Meanwhile, the release of curcumin solution was also examined so as to confirm whether the dialysis mem- of EE. The reasons for the variety in EE at different brane would affect curcumin diffusion. As shown in pH values are not very clear but may be related to Fig. 5a, curcumin was released very fast from its solu- the solubility of curcumin which is soluble in alkali tion (>80% at 6 h), indicating that the dialysis bag had or extremely acidic solvents [33]. no effect on curcumin diffusion. In contrast to the The morphology of liposomes examined by SEM is rapid release of curcumin solution, all the Cur-LPs shown in Fig. 3. The particles of LP-2.5 (Fig. 3a) and showed an obvious sustained release property (Fig. 5b), LP-5.0 (Fig. 3b) are spherical in shape and have a uniform and the release profiles were very similar to each other, Shao et al. Nanoscale Research Letters (2017) 12:504 Page 5 of 8 Fig. 4 Physical stability of liposomes with varied micro-environmental pH values (pH 2.5, 5.0, and 7.4). a Thermodynamic stability indicating particles aggregation. b Kinetic stability indicating particle sedimentation. The data is presented as mean ± sd (n =3) around 5%). After 8 h, curcumin was released a little slower and the cumulative release percentage was ~30% within 72 h. It is assumed that the release speed in vivo or in the presence of serum would be substantially faster due partially to the metabolism of lipid. Interestingly, the release profiles of all the three Cur- LPs are close to straight lines. Therefore, the linear fitting to the three release profiles was performed. As shown in Table 1, all these profiles showed very good linearity with fitting degree higher than 0.99 (regression equations are also displayed), suggesting that the release of Cur-LPs fitted to zero-order kinetics. In other similar studies, the release of curcumin from liposome was found to be non-linear [38, 39]. From the point view of Fig. 3 SEM images of liposome with micro-environmental pH of a drug research and development, zero-order release 2.5, b 5.0, and c 7.4. Scale bar,1 μm kinetics is the most ideal release profile because it pro- vides a constant drug release rate and thus is able to indicating that micro-environmental pH had no significant maintain therapeutic effect for a long time, decrease effect on curcumin release speed. In detail, curcumin was administration times, and reduce side effects. There- released a little faster in the first 8 h due probably to the fore, the LPs prepared in this work may be promising initial burst release (the cumulative release percentage was carriers for controlled drug delivery. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 6 of 8 Fig. 6 Chemical stability of liposomal curcumin (Cur-LPs) with varied micro-environmental pH values (pH 2.5, 5.0, and 7.4). The stability was examined by quantifying the remained curcumin after incubating Cur-LPs with 50% FBS. The data is presented as mean ± sd (n =3). Statistical significance between groups: ***p < 0.001, **p <0.01, *p <0.05 LP-5.0 > Cur-LP-7.4. This pH-dependent chemical stability of Cur-LP is consistent with another work, which showed the pH-dependent stability of free curcu- min [26]. The in vitro release was performed in serum- free medium, and the cumulative release could be 30% at 72 h. However, the chemical stability study was performed in serum-containing solution, in which serum enzymes could degrade the released curcumin and also Fig. 5 In vitro release profiles of different curcumin formulations in PBS (pH 7.4). a Curcumin solution, in which curcumin was dissolved break liposome and thus degrade the unreleased curcu- in PBS containing 0.1% Tween 80 (pH 7.4). b Cur-LPs with varied min. This is the reason why 30% curcumin was released at micro-environmental pH of 2.5, 5.0, and 7.4, respectively. The data 72 h in the in vitro release study but only 55% remained at is presented as mean ± sd (n =3) 6 h for Cur-LP-2.5 in the serum stability study. Liposomes consist of two parts in structure: one is the Effect of Micro-environmental pH on Chemical Stability hydrophobic lipid bilayer and the other is the hydro- of Cur-LP philic inner aqueous chamber. It is easy to understand The chemical stability of liposomal curcumin in FBS is that a pH-sensitive hydrophilic drug would be located in showninFig.6.After incubation for2h, 89% curcumin the inner aqueous chamber, and its stability would be remained for Cur-LP-2.5, significantly higher than 74% significantly affected by the micro-environmental pH in for Cur-LP-5.0 and 61% for Cur-LP-7.4 (p <0.001). At the aqueous chamber, where the buffering volume and 4 h post-incubation, 69% curcumin remained for Cur- buffering capacity would be much higher than that in LP-2.5, significantly higher than 53% for Cur-LP-5.0 the lipid bilayer. In contrast, curcumin is a hydrophobic and 40% for Cur-LP-7.4 (p < 0.01). At 6 h post-incubation, molecule and would be located in the lipid bilayer. For 55% curcumin remained for Cur-LP-2.5, still significantly this reason, it is quite interesting to find the micro- higher than 43% for Cur-LP-5.0 and 34% for Cur-LP-7.4 environmental pH-dependent chemical stability of lipo- (p < 0.05). It is clear that the chemical stability of Cur-LPs somal curcumin. It is assumed that the space in lipid is micro-environmental pH-dependent: Cur-LP-2.5 > Cur- bilayer would not be absolutely anhydrous although it is hydrophobic. As we know, the living cell membrane is not absolutely anhydrous in its lipid bilayer. Instead, it Table 1 Linear fitting of the in vitro release profiles of Cur-LPs contains a certain small volume of aqueous solution for with varied micro-environmental pH values transport of water-soluble molecules and ions. Likewise, Liposomes Regression equation Fitting degree (r) a certain small volume of buffer solution with the same Cur-LP-2.5 y = 0.424x + 0.608 0.9985 components as the inner chamber would also exist in Cur-LP-5.0 y = 0.403x + 0.800 0.9975 the hydrophobic lipid bilayer after successful preparation Cur-LP-7.4 y = 0.449x + 0.885 0.9980 of liposome. Thus, the hydrophobic drug located in the Shao et al. Nanoscale Research Letters (2017) 12:504 Page 7 of 8 lipid bilayer can be directly affected by the micro- a micro-environmental pH-dependent manner. After environmental pH of liposome. In addition, the acidic treatment for 1 day, Cur-LP-2.5 and Cur-LP-5.0 showed micro-environment may reduce the activities of some a significantly stronger ability to inhibit HepG2 cell enzymes which show the best activity in normal physio- growth than Cur-LP-7.4 (the cell viability was 80% for logical condition. This also contributes to the higher Cur-LP-2.5 and Cur-LP-5.0, and 90% for Cur-LP-7.4). At chemical stability of liposomal curcumin in the lower day 3 post-treatment, the cell viability decreased substan- micro-environmental pH. It has been reported that lipo- tially, and Cur-LP-2.5 and Cur-LP-5.0 showed comparable somes composed of egg phosphatidylcholine (EPC) anticancer efficacy and significantly higher than Cur-LP- rapidly lost their internal pH-gradient in buffer (pH 7.4), 7.4. The cell viability was 24% for Cur-LP-2.5 (p <0.05 vs and the pH-gradient maintaining ability was substan- Cur-LP-7.4), 21% for Cur-LP-5.0 (p < 0.01 vs Cur-LP-7.4), tially enhanced by substituting EPC (phase transition and 39% for Cur-LP-7.4. These results indicate that the temperature (T ) ≈−5 °C) with the high T (41 °C) lipid anticancer efficacy of liposomal curcumin is micro- m m DPPC (dipalmitoyl phosphatidylcholine) and by addition environmental pH- and time-dependent. In consideration of cholesterol [40]. In this present work, the liposome is of the higher EE and chemical stability of Cur-LP-2.5 than composed of soybean lecithin (T is around 238.2 °C Cur-LP-5.0, liposome with micro-environmental pH of 2.5 [41]) and cholesterol. Hence, the micro-environmental would have the greatest potential for practical application. pH-gradient of liposomes prepared in this work can be expected to maintain for a long period. This is a strong support to the results and assumptions shown above. Conclusions Liposome, as a widely used drug delivery system, is cap- In Vitro Anticancer Efficacy able of improving the solubility of water-insoluble drugs, We have demonstrated the micro-environmental pH- protecting the drug payloads from the harsh physio- dependent chemical stability of liposomal curcumin logical environment and delivering the payloads to a above. Here, we conducted a preliminary in vitro study targeted tissue. However, the delivery of pH-sensitive to investigate the anticancer efficacy of these liposomal drugs is still limited by their natural instability in physio- curcumin. Interestingly, the blank LPs could enhance logical conditions (neutral environment). In this present the cell growth at day 1 and maintain this function to work, we propose a novel approach for enhancing the some extent till day 3 in comparison to the control chemical stability of pH-sensitive drug payloads by regu- group (Fig. 7). This indicates that blank LPs may provide lating the micro-environmental acidity of liposome. The nutrition to cells, which is consistent with our previous findings show that the chemical stability and in vitro report [27]. The free curcumin showed little anticancer efficacy of the model pH-sensitive drug curcumin is sig- efficacy due to its quite limited solubility. In contrast, nificantly enhanced by acidifying the micro-environment of Cur-LPs demonstrated significant anticancer efficacy in liposome. In conclusion, regulation of micro-environmental pH of liposome is feasible to enhance the chemical stability of pH-sensitive drug payloads, even for the hydrophobic drugs which are located in the lipid bilayer. Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 81402860, 81470721), the Excellent Young Scientist Foundation of Sichuan University to Q. Peng (no. 2082604194312), and Sichuan Science and Technology Innovation Team (no. 2014TD0001). Authors’ Contributions XRS and XQW performed the experiments and collected the data. SZ and NF performed the experiments. YFL and XXC analyzed the data. QP conceived and designed the study, analyzed the data, and wrote the paper. All authors read and approved the final manuscript. Fig. 7 Anticancer efficacy of liposomal curcumin with varied micro- Competing Interests environmental pH (2.5, 5.0, and 7.4). The viability of HepG2 cells at The authors declare that they have no competing interests. days 1 and 3 after treatment by blank LPs, free curcumin, and Cur-LPs was examined by cck-8 assay. The cells treated by serum-contained blank culture medium served as control. The data is presented Publisher’sNote as mean ± sd (n = 3). Statistical significance between groups: Springer Nature remains neutral with regard to jurisdictional claims in **p < 0.01, *p <0.05 published maps and institutional affiliations. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 8 of 8 Received: 8 June 2017 Accepted: 28 July 2017 25. Mehanny M, Hathout RM, Geneidi AS, Mansour S (2016) Exploring the use of nanocarrier systems to deliver the magical molecule; curcumin and its derivatives. J Control Release 225:1–30 26. 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Effects of Micro-environmental pH of Liposome on Chemical Stability of Loaded Drug

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Springer Journals
Copyright
Copyright © 2017 by The Author(s).
Subject
Materials Science; Nanotechnology; Nanotechnology and Microengineering; Nanoscale Science and Technology; Nanochemistry; Molecular Medicine
ISSN
1931-7573
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1556-276X
DOI
10.1186/s11671-017-2256-9
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Abstract

Liposome is a promising carrier system for delivering bioactive molecules. However, the successful delivery of pH-sensitive molecules is still limited by the intrinsic instability of payloads in physiological environment. Herein, we developed a special liposome system that possesses an acidic micro-environment in the internal aqueous chamber to improve the chemical stability of pH-sensitive payloads. Curcumin-loaded liposomes (Cur-LPs) with varied internal pH values (pH 2.5, 5.0, or 7.4) were prepared. These Cur-LPs have similar particle size of 300 nm, comparable physical stabilities and analogous in vitro release profiles. Interestingly, the chemical stability of liposomal curcumin in 50% fetal bovine serum and its anticancer efficacy in vitro are both micro-environmental pH-dependent (Cur-LP-2.5 > Cur-LP-5.0 > Cur-LP-7.4). This serum stability still has space to be further enhanced to improve the applicability of Cur-LP. In conclusion, creating an acidic micro-environment in the internal chamber of liposome is feasible and efficient to improve the chemical stability of pH-sensitive payloads. Keywords: Liposomes, Nanoparticles, Drug delivery, Controlled release, Curcumin Background values). Generally, liposome is prepared in a neutral buffer Liposome, an artificial membrane vehicle, has shown solution and thus the loaded drug molecules are also in a great potentials in drug delivery due to its drug loading neutral environment after incorporation into liposome. capacity, biodegradability, and biocompatibility [1–4]. Accordingly, those molecules which are only stable in The classic liposome is similar with living cells in struc- acidic environment would be still instable even in the ture, typically consisting of a phospholipid bilayer and form of liposome. Therefore, development of a novel an aqueous inner chamber [5–7]. Due to this structure, approach for enhancing the stability of pH-sensitive drugs liposome is able to solubilize the insoluble drug molecules is of great importance for successful delivery of these and prevent the loaded drug from the harsh physiological payloads by liposome. environment [8–10]. In addition, the surface of liposome As mentioned above, liposome has an aqueous space can be modified to prolong the blood circulation time in its inner chamber, which can be used to provide drug and/or target specific tissues [11–15]. With these above- payloads with an acidic micro-environment (Fig. 1). In mentioned advantages, various liposome systems have this present work, we use curcumin as a model drug and been clinically approved [8, 9, 16]. aim to provide a novel approach for enhancing the Although the delivery of many drugs has been improved chemical stability of drug molecules loaded in liposome. by incorporation into liposome, the delivery of some It is well known that curcumin is a lipophilic molecule pH-sensitive drugs is still limited by the instability of drug and has been extensively used in food, medicines, and molecule itself in physiological environment (neutral pH cosmetics due to its various bioactivities [17–21]. How- ever, its delivery is highly limited by its insolubility and * Correspondence: dentistcai@hotmail.com; qiangpengzz@scu.edu.cn; instability in biological fluids [22–25]. So far, it is yet to lijm2002@163.com fulfill its clinical promise in part due to pH-mediated Equal contributors instability [26]. Therefore, curcumin is a suitable model State Key Laboratory of Oral Diseases, West China Hospital of Stomatology, Sichuan University, No. 14, Block 3, Renmin Road South, Chengdu 610041, drug for this work. China © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 2 of 8 Fig. 1 Schematics of the liposome with varied micro-environmental acidity in its inner aqueous chamber Methods The size and zeta potential of LP were determined by Materials dynamic light scattering (DLS) and electrophoretic light Phospholipids (soybean lecithin for injection use) were scattering (ELS), respectively, using ZetasizerNano ZS90 purchased from Shanghai Tai-Wei Pharmaceutical Co., (Malvern Instruments Ltd., Malvern, UK) at 25 °C [29]. Ltd., (Shanghai, China). Cholesterol was obtained from The measurement cycle was automatically determined by Amresco (Solon, OH, USA). Poloxamer 188 (F68) was the instrument system. The particle size was presented by kindly donated by BASF (China) Co., Ltd., (Shanghai, intensity distribution, and the size distribution was evalu- China). Curcumin was supplied by Sigma (St. Louis, MO, ated by polydispersity index (PDI). USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA). All other chemical reagents Encapsulation Efficiency (EE) Determination used in this study were of analytical grade or better. EE, an important parameter for quality control, is of great significance in developing liposome-based delivery Preparation of Curcumin-Loaded Liposomes (Cur-LPs) systems. The EE determination was based on the high The liposomes with varied micro-environmental pH values speed centrifugation method. Briefly, 100 μl Cur-LPs were prepared using evaporation method according to pre- was centrifuged at the low speed (3000 rpm, 5 min) to vious works with some modifications [27, 28]. Briefly, precipitate non-dissolved free curcumin, and 50 μl phospholipids (75 mg) and cholesterol (5 mg) were dis- supernatant was subjected to high-speed centrifugation solved in 0.5 ml ethanol containing 2 mg/ml curcumin. (16 krpm, 10 min) to separate Cur-LPs from the tiny The ethanol solution was mixed with 5 ml 0.001 M PBS dissolved curcumin. The pellets were re-suspended in containing 1% (w/v) F68 that served as a surfactant to 500 μl PBS (i.e., 10-fold dilution), an aliquot of 10 μlof narrow the size distribution. After magnetically stirring for which was mixed with 300 μl ethanol by vortex and son- 1 min (constant temperature magnetic mixer, DF-101S, ication for 30 s. The fluorescent intensity of curcumin in Zhengzhou Greatwall Scientific Industrial and Trade Co., the resultant solution was determined (excitation wave- Ltd., Zhengzhou, China), the resultant emulsion was evap- length (Ex), 458 nm; emission wavelength (Em), 548 nm) orated under vacuum and dark for 30 min at 35 °C to and presented as F , i.e., the fluorescent intensity of remove ethanol. The acidity in the inner chamber of Cur- encapsulated curcumin. Another 50 μl of fresh Cur-LP LP was adjusted via using PBS with varied pH values of containing encapsulated and free curcumin was also 2.5, 5.0, or 7.4 during preparation. The resultant suspen- diluted by 10-fold with PBS, and 10 μl of the diluted sion was centrifuged at a low speed (3000 rpm, 5 min) to solution were mixed with 300 μl ethanol. The fluores- precipitate free curcumin. The supernatant was then cen- cent intensity of the resultant solution was measured trifuged at a high speed (16 krpm, 10 min), and the pellets and presented as F , i.e., the fluorescent intensity of total were re-suspended in PBS (pH 7.4) before further use. curcumin. The EE was therefore calculated with the This procedure provided these LPs with an identical exter- following equation: EE = F /F . e t nal environment. The obtained liposomes with different micro-environmental pH values were presented as Cur- Scanning Electron Microscopy (SEM) LP-2.5, Cur-LP-5.0, and Cur-LP-7.4, respectively. Blank The morphology of LP was observed by the scanning elec- liposomes were also fabricated as above. tron microscopy (SEM, INSPECT F, FEI, Netherlands) [30]. Briefly, the LP suspension was 100-fold diluted with Characterization of Liposome distilled water, and one drop of the diluted suspension was The hydrodynamic size, size distribution, and zeta poten- placed on a clean glass sheet. After air-drying, the sample tial are the three basic parameters for liposome systems. was coated with gold right before SEM. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 3 of 8 Physical Stability of Liposomes onto the 96-well cell culture plates at a density of 10,000 Physical stability is a very meaningful parameter for stor- cells per well and cultured under standard conditions age and transportation of a colloidal system. The phys- (37 °C/5% CO ) for 24 h in PRIM-1640 culture medium ical stability of liposome was presented by colloidal supplemented with 10% FBS. Subsequently, the culture stability and investigated according to a previous method medium was removed and cells were washed with PBS. [31]. Briefly, 100 μl of LP was added to tubes and kept at The Cur-LPs were diluted in serum-free culture medium 37 °C. At different time intervals, the LP size was mea- (4 μg/ml curcumin) and added to cells, followed by sured and compared to the initial size so as to indicate continuous incubation for 1 and 3 days at 37 °C. The the thermodynamic stability. In addition, another 300 μl OD value of viable cells was measured by cck-8 assay. of LP were also added to tubes and kept at 37 °C. At the The cells treated with blank culture medium served as same time intervals, 100 μl of the upper layer liquid control and cell viability (%) was the OD value percent- were collected. The transmittance of the collected speci- age of specimens relative to control. mens was measured at 550 nm and compared to the initial value so as to indicate the kinetic stability. Statistics All the data are presented as mean ± sd (standard devi- In Vitro Release ations). The differences between two groups, analyzed The release profile of liposome plays an important role by the Student’s t test, were considered to be statisti- in predicting in vivo fate and efficacy of liposome. The cally significant when the p value was less than 0.05. in vitro release of curcumin from Cur-LP was studied using the dynamic dialysis method [32]. Briefly, 1 ml of Results and Discussion each Cur-LP was added into a dialysis bag (molecular Characterization of Liposome weight cutoff, 10 kD), which was used to retain liposome The micro-environmental pH of liposome refers to but keep the released curcumin molecules permeable. theacidity in theinner aqueouschamber of liposome The specimen-loaded dialysis bag was soaked in 4 ml (Fig. 1), which is different from the pH in the exter- release medium (0.001 M PBS containing 0.1% Tween nal environment. In this work, the external environ- 80, pH 7.4), and the release study was conducted away mental pH of all liposome suspensions was 7.4 unless from light (37 °C, 100 rpm). At each fixed time interval, otherwise stated. the release medium was collected and replaced with Particle size, zeta potential, and encapsulation effi- 4 ml fresh medium so as to simulate sink conditions. ciency (EE) are important parameters for quality con- The collected medium was diluted to 5 ml with PBS and trol of liposome. The size of three Cur-LPs was similar further diluted by 15-fold with ethanol. The curcumin in to each other (around 300 nm, Fig. 2a). The PDI of the resultant solution was quantified by fluorescence each formulation was lower than 0.2, indicating a nar- spectrophotometry (Ex 458 nm, Em 548 nm). In row size distribution. Interestingly, the negative zeta addition, curcumin powder was dissolved in the above potential of Cur-LP-7.4 (−9 mV) is significantly lower release medium, and the release of curcumin solution than that of the other two Cur-LPs (~−18 mV). Usually, was conducted at pH 7.4 to investigate whether the the negative zeta potential would decrease and even dialysis bag would retain curcumin molecules. convert to positive value with decreasing the pH of disperse phase due to the increase of H concentration. Chemical Stability of Liposomal Curcumin We indeed observed this phenomenon when preparing Chemical stability is a key parameter for predicting drug Cur-LPs in non-buffer HCl/NaOH solutions with pH of metabolism, efficacy, and toxicity. The chemical stability 2.5, 5.0, and 7.4 (Fig. 2b). In the case of PBS, however, 3− 2− − of Cur-LPs was examined in 50% FBS. Briefly, 100 μlof the existence of PO ,HPO ,and/or H PO and their 4 4 2 4 Cur-LPs were diluted by 10-fold with PBS (pH 7.4) and interaction with LP may lead to more complicated situ- then mixed with 1 ml FBS. The specimens were shaken on ations and different results. It is well recognized that a horizontal shaker away from light (37 °C, 100 rpm). At zeta potential plays key roles in maintaining the fixed time intervals, an aliquot of 10 μl of specimen was colloidal stability of nano-scaled suspension. In general, collected and mixed with 300 μl ethanol immediately a higher absolute value of zeta potential leads to a more followed by centrifugation (16 krpm, 5 min). The remained stable colloidal suspension system. curcumin in the supernatant was quantified as above. EE is of concern during liposome development. Usually, increasing EE is important for reducing cost and enhan- In Vitro Anticancer Efficacy cing efficacy. In this work, the EE of Cur-LP-2.5 is 74% The preliminary anticancer efficacy of the three Cur-LPs (Fig. 2c), which is the highest among Cur-LP-5.0 (45%) was investigated using human liver hepatocellular and Cur-LP-7.4 (64%), indicating that Cur-LP-2.5 is the carcinoma HepG2 cells. Briefly, HepG2 cells were plated best formulation for delivering curcumin from the point Shao et al. Nanoscale Research Letters (2017) 12:504 Page 4 of 8 particle distribution. The LP-7.4 also shows a sphere-like shape, but adhesion among particles can be clearly observed (Fig. 3c), indicating that the drying process during SEM specimen preparation would lead to aggre- gation of LP-7.4. This may be due to the relatively low absolute value of zeta potential of LP-7.4 (Fig. 2a). Add- itionally, the particle size measured by SEM is smaller than the hydrodynamic size measured by DLS, which is due to the loss of hydration shell of liposome after drying process for SEM. Physical Stability of Liposome Liposome is a colloidal system and its physical stability can be presented by colloidal stability, which has sub- stantial impacts on liposome storage and further applica- tions [34, 35]. Particle aggregation (thermodynamic instability) and sedimentation (kinetic instability) are the two essential aspects of colloidal instability. Aggregation leads to a larger apparent size and sedimentation leads to changes in transmittance of suspension. More import- antly, size increase can directly affect the efficacy of nano-systems since particle size has been shown to have great impacts on cellular uptake, cytotoxicity, pharmaco- kinetic profile, and tissue distribution [36, 37]. Here, we examined the aggregation and sedimenta- tion properties of three liposome systems to indicate their thermodynamic and kinetic stability, respectively. As shown in Fig. 4a, the three LPs showed no substan- tial changes in hydrodynamic size within 72 h, indicat- ing that all these LPs have a very high thermodynamic stability. Meanwhile, the transmittance change of all the three LPs was less than 10% (Fig. 4b), indicating little particle sedimentation and thus a high kinetic stability. These results suggestthatthe threeLPs have an excellent colloidal stability within 72 h, and the micro-environmental pH has no influence on physical stability of liposome. In Vitro Release Drug release profile from liposome is usually examined to evaluate formulation quality, provide reference for Fig. 2 Physicochemical characterization of liposomes. a Hydrodynamic size and zeta potential of Cur-LPs fabricated in PBS with pH 2.5, 5.0, dosage regimen, and predict the effectiveness in vivo. In and 7.4, respectively. b Zeta potential of Cur-LPs fabricated in HCl/ general, almost all liposomal systems have a sustained NaOH solutions with pH 2.5, 5.0, and 7.4, respectively. c Encapsulation drug release property. Here, we examined the in vitro efficiency of Cur-LPs prepared in PBS. Data presented as mean ± sd release behavior of three Cur-LPs in PBS (pH 7.4). (n = 3). Statistical significance between groups: ***p <0.001 Meanwhile, the release of curcumin solution was also examined so as to confirm whether the dialysis mem- of EE. The reasons for the variety in EE at different brane would affect curcumin diffusion. As shown in pH values are not very clear but may be related to Fig. 5a, curcumin was released very fast from its solu- the solubility of curcumin which is soluble in alkali tion (>80% at 6 h), indicating that the dialysis bag had or extremely acidic solvents [33]. no effect on curcumin diffusion. In contrast to the The morphology of liposomes examined by SEM is rapid release of curcumin solution, all the Cur-LPs shown in Fig. 3. The particles of LP-2.5 (Fig. 3a) and showed an obvious sustained release property (Fig. 5b), LP-5.0 (Fig. 3b) are spherical in shape and have a uniform and the release profiles were very similar to each other, Shao et al. Nanoscale Research Letters (2017) 12:504 Page 5 of 8 Fig. 4 Physical stability of liposomes with varied micro-environmental pH values (pH 2.5, 5.0, and 7.4). a Thermodynamic stability indicating particles aggregation. b Kinetic stability indicating particle sedimentation. The data is presented as mean ± sd (n =3) around 5%). After 8 h, curcumin was released a little slower and the cumulative release percentage was ~30% within 72 h. It is assumed that the release speed in vivo or in the presence of serum would be substantially faster due partially to the metabolism of lipid. Interestingly, the release profiles of all the three Cur- LPs are close to straight lines. Therefore, the linear fitting to the three release profiles was performed. As shown in Table 1, all these profiles showed very good linearity with fitting degree higher than 0.99 (regression equations are also displayed), suggesting that the release of Cur-LPs fitted to zero-order kinetics. In other similar studies, the release of curcumin from liposome was found to be non-linear [38, 39]. From the point view of Fig. 3 SEM images of liposome with micro-environmental pH of a drug research and development, zero-order release 2.5, b 5.0, and c 7.4. Scale bar,1 μm kinetics is the most ideal release profile because it pro- vides a constant drug release rate and thus is able to indicating that micro-environmental pH had no significant maintain therapeutic effect for a long time, decrease effect on curcumin release speed. In detail, curcumin was administration times, and reduce side effects. There- released a little faster in the first 8 h due probably to the fore, the LPs prepared in this work may be promising initial burst release (the cumulative release percentage was carriers for controlled drug delivery. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 6 of 8 Fig. 6 Chemical stability of liposomal curcumin (Cur-LPs) with varied micro-environmental pH values (pH 2.5, 5.0, and 7.4). The stability was examined by quantifying the remained curcumin after incubating Cur-LPs with 50% FBS. The data is presented as mean ± sd (n =3). Statistical significance between groups: ***p < 0.001, **p <0.01, *p <0.05 LP-5.0 > Cur-LP-7.4. This pH-dependent chemical stability of Cur-LP is consistent with another work, which showed the pH-dependent stability of free curcu- min [26]. The in vitro release was performed in serum- free medium, and the cumulative release could be 30% at 72 h. However, the chemical stability study was performed in serum-containing solution, in which serum enzymes could degrade the released curcumin and also Fig. 5 In vitro release profiles of different curcumin formulations in PBS (pH 7.4). a Curcumin solution, in which curcumin was dissolved break liposome and thus degrade the unreleased curcu- in PBS containing 0.1% Tween 80 (pH 7.4). b Cur-LPs with varied min. This is the reason why 30% curcumin was released at micro-environmental pH of 2.5, 5.0, and 7.4, respectively. The data 72 h in the in vitro release study but only 55% remained at is presented as mean ± sd (n =3) 6 h for Cur-LP-2.5 in the serum stability study. Liposomes consist of two parts in structure: one is the Effect of Micro-environmental pH on Chemical Stability hydrophobic lipid bilayer and the other is the hydro- of Cur-LP philic inner aqueous chamber. It is easy to understand The chemical stability of liposomal curcumin in FBS is that a pH-sensitive hydrophilic drug would be located in showninFig.6.After incubation for2h, 89% curcumin the inner aqueous chamber, and its stability would be remained for Cur-LP-2.5, significantly higher than 74% significantly affected by the micro-environmental pH in for Cur-LP-5.0 and 61% for Cur-LP-7.4 (p <0.001). At the aqueous chamber, where the buffering volume and 4 h post-incubation, 69% curcumin remained for Cur- buffering capacity would be much higher than that in LP-2.5, significantly higher than 53% for Cur-LP-5.0 the lipid bilayer. In contrast, curcumin is a hydrophobic and 40% for Cur-LP-7.4 (p < 0.01). At 6 h post-incubation, molecule and would be located in the lipid bilayer. For 55% curcumin remained for Cur-LP-2.5, still significantly this reason, it is quite interesting to find the micro- higher than 43% for Cur-LP-5.0 and 34% for Cur-LP-7.4 environmental pH-dependent chemical stability of lipo- (p < 0.05). It is clear that the chemical stability of Cur-LPs somal curcumin. It is assumed that the space in lipid is micro-environmental pH-dependent: Cur-LP-2.5 > Cur- bilayer would not be absolutely anhydrous although it is hydrophobic. As we know, the living cell membrane is not absolutely anhydrous in its lipid bilayer. Instead, it Table 1 Linear fitting of the in vitro release profiles of Cur-LPs contains a certain small volume of aqueous solution for with varied micro-environmental pH values transport of water-soluble molecules and ions. Likewise, Liposomes Regression equation Fitting degree (r) a certain small volume of buffer solution with the same Cur-LP-2.5 y = 0.424x + 0.608 0.9985 components as the inner chamber would also exist in Cur-LP-5.0 y = 0.403x + 0.800 0.9975 the hydrophobic lipid bilayer after successful preparation Cur-LP-7.4 y = 0.449x + 0.885 0.9980 of liposome. Thus, the hydrophobic drug located in the Shao et al. Nanoscale Research Letters (2017) 12:504 Page 7 of 8 lipid bilayer can be directly affected by the micro- a micro-environmental pH-dependent manner. After environmental pH of liposome. In addition, the acidic treatment for 1 day, Cur-LP-2.5 and Cur-LP-5.0 showed micro-environment may reduce the activities of some a significantly stronger ability to inhibit HepG2 cell enzymes which show the best activity in normal physio- growth than Cur-LP-7.4 (the cell viability was 80% for logical condition. This also contributes to the higher Cur-LP-2.5 and Cur-LP-5.0, and 90% for Cur-LP-7.4). At chemical stability of liposomal curcumin in the lower day 3 post-treatment, the cell viability decreased substan- micro-environmental pH. It has been reported that lipo- tially, and Cur-LP-2.5 and Cur-LP-5.0 showed comparable somes composed of egg phosphatidylcholine (EPC) anticancer efficacy and significantly higher than Cur-LP- rapidly lost their internal pH-gradient in buffer (pH 7.4), 7.4. The cell viability was 24% for Cur-LP-2.5 (p <0.05 vs and the pH-gradient maintaining ability was substan- Cur-LP-7.4), 21% for Cur-LP-5.0 (p < 0.01 vs Cur-LP-7.4), tially enhanced by substituting EPC (phase transition and 39% for Cur-LP-7.4. These results indicate that the temperature (T ) ≈−5 °C) with the high T (41 °C) lipid anticancer efficacy of liposomal curcumin is micro- m m DPPC (dipalmitoyl phosphatidylcholine) and by addition environmental pH- and time-dependent. In consideration of cholesterol [40]. In this present work, the liposome is of the higher EE and chemical stability of Cur-LP-2.5 than composed of soybean lecithin (T is around 238.2 °C Cur-LP-5.0, liposome with micro-environmental pH of 2.5 [41]) and cholesterol. Hence, the micro-environmental would have the greatest potential for practical application. pH-gradient of liposomes prepared in this work can be expected to maintain for a long period. This is a strong support to the results and assumptions shown above. Conclusions Liposome, as a widely used drug delivery system, is cap- In Vitro Anticancer Efficacy able of improving the solubility of water-insoluble drugs, We have demonstrated the micro-environmental pH- protecting the drug payloads from the harsh physio- dependent chemical stability of liposomal curcumin logical environment and delivering the payloads to a above. Here, we conducted a preliminary in vitro study targeted tissue. However, the delivery of pH-sensitive to investigate the anticancer efficacy of these liposomal drugs is still limited by their natural instability in physio- curcumin. Interestingly, the blank LPs could enhance logical conditions (neutral environment). In this present the cell growth at day 1 and maintain this function to work, we propose a novel approach for enhancing the some extent till day 3 in comparison to the control chemical stability of pH-sensitive drug payloads by regu- group (Fig. 7). This indicates that blank LPs may provide lating the micro-environmental acidity of liposome. The nutrition to cells, which is consistent with our previous findings show that the chemical stability and in vitro report [27]. The free curcumin showed little anticancer efficacy of the model pH-sensitive drug curcumin is sig- efficacy due to its quite limited solubility. In contrast, nificantly enhanced by acidifying the micro-environment of Cur-LPs demonstrated significant anticancer efficacy in liposome. In conclusion, regulation of micro-environmental pH of liposome is feasible to enhance the chemical stability of pH-sensitive drug payloads, even for the hydrophobic drugs which are located in the lipid bilayer. Acknowledgements This work was supported by the National Natural Science Foundation of China (nos. 81402860, 81470721), the Excellent Young Scientist Foundation of Sichuan University to Q. Peng (no. 2082604194312), and Sichuan Science and Technology Innovation Team (no. 2014TD0001). Authors’ Contributions XRS and XQW performed the experiments and collected the data. SZ and NF performed the experiments. YFL and XXC analyzed the data. QP conceived and designed the study, analyzed the data, and wrote the paper. All authors read and approved the final manuscript. Fig. 7 Anticancer efficacy of liposomal curcumin with varied micro- Competing Interests environmental pH (2.5, 5.0, and 7.4). The viability of HepG2 cells at The authors declare that they have no competing interests. days 1 and 3 after treatment by blank LPs, free curcumin, and Cur-LPs was examined by cck-8 assay. The cells treated by serum-contained blank culture medium served as control. The data is presented Publisher’sNote as mean ± sd (n = 3). Statistical significance between groups: Springer Nature remains neutral with regard to jurisdictional claims in **p < 0.01, *p <0.05 published maps and institutional affiliations. Shao et al. Nanoscale Research Letters (2017) 12:504 Page 8 of 8 Received: 8 June 2017 Accepted: 28 July 2017 25. Mehanny M, Hathout RM, Geneidi AS, Mansour S (2016) Exploring the use of nanocarrier systems to deliver the magical molecule; curcumin and its derivatives. J Control Release 225:1–30 26. 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Published: Aug 23, 2017

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