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Pharmaceutical excipient salts effect on micellization and drug solubilization of PEO-PPO-ph-PPO-PEO block copolymer
Colloids and Surfaces B: Biointerfaces 189 (2020) 110857
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Pharmaceutical excipient salts effect on micellization and drug solubilization of PEO-PPO-ph-PPO-PEO block copolymer
T
Yaoyao Zhua,b, Mengran Chua,b, Zixiao Wanga,b, Yutao Xuea, Bo Liua, Jie Suna, Teng Liua,* a b
Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, PR China School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: PEO-PPO copolymer Hydrophobic modification Sodium citrate Micelles Drug solubilization
Hydrophobic modification PEO-PPO copolymer BP123 was synthesized, with two aromatic rings in the centre linked to PEO-PPO blocks, and the identical PEO and PPO block numbers were possessed with commercial copolymer P123. The influence of three common pharmaceutical excipient salts sodium chloride (NaCl), sodium citrate (NaCA) and sodium benzoate (NaBZ) on self-assembly behaviors of BP123 and P123 was investigated via cloud point, surface tension, pyrene fluorescence and dynamic light scattering. Solubilization for hydrophobic drug simvastatin (SV) and in vitro drug release behavior were assessed accordingly. In the presence of NaCl or NaCA, cloud point and critical micellization concentration (CMC) decreased, micelles became more hydrophobic, micellar size and drug solubilization increased, drug release rate slowed, and the impact of NaCA was more significant than NaCl. Oppositely, cloud point and CMC increased with the addition of NaBZ. NaBZ could participate in the formation of micelles by hydrophobic aromatic ring, which greatly raised solubilization of SV. Moreover, a different performance occurred when NaBZ was added to BP123 or P123, due to the hydrophobic benzene rings in BP123, which prominently enhanced the interaction with hydrophobic drug, leading to obvious delay of drug release for BP123. This work is conducive to turning copolymer property in diverse pharmaceutical applications and in drug delivery systems as drug carriers.
1. Introduction Block copolymers comprise of poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) blocks and are commercially available under the trade name Pluronic (PEO-PPO-PEO), which have been extensively studied from viewpoint of aqueous solution behaviors [1–3]. They selfassemble to form micelles in aqueous solutions, with PPO groups forming a fairly compact core and hydrated PEO groups constituting a swollen protective corona, and these core-shell micelles could effectively solubilize hydrophobic drugs. Pluronic copolymers are biocompatible, which have remarkable applications in controlled drug encapsulation systems since their micelles own a slower rate of dissociation and allow the preservation of loaded drugs for a longer period of time [4,5]. The aggregation behavior and solubilizing ability of Pluronic depend on various factors, such as copolymer structure, ionic strength and different additives. Chemical nature of copolymer obviously affects aggregation behavior and drug load ability. Molecular characteristics, such as PPO/PEO ratio and molecular weight, have been widely investigated [6,7]. When
Pluronic contains same PEO segments, critical micellization concentration (CMC) decreases as PPO content increases. Pluronic with higher PPO/PEO ratio possesses larger micelles and more hydrophobic cores, so its solubilization ability is better. Recently, many research groups have studied the influence of hydrophobic modification on the self-assemble process of copolymers [8,9]. The existence of hydrophobic groups in copolymer could notably raise the adsorption ability and strengthen aggregation behavior. Nevertheless, researches about hydrophobic modification of PEO-PPO copolymers are just rising and there remains much unknown information. Ionic strength clearly impacts the aggregation behavior of Pluronic. The classical order for common inorganic salt ions to influence the aggregation behaviors of copolymers is basically determined, according to Hofmeister series generally [10,11]. On the one hand, some salt ions are manifested as "salting out" effect and water structure makers, including K+, Na+, SO42−, Cl-, Br-, are characterized by weakening the hydration of copolymers as both CMC and cloud point decrease. Simultaneously, these ions can enhance the solubilization of hydrophobic drug, because they remove water from the core and improve the
⁎ Corresponding author at: Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jingshi Road No.18877, Jinan, 250062, PR China. E-mail address: [email protected] (T. Liu).
https://doi.org/10.1016/j.colsurfb.2020.110857 Received 19 October 2019; Received in revised form 14 January 2020; Accepted 10 February 2020 Available online 10 February 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Cloud point
hydrophobicity, as well as make more room to load drug [12,13]. On the other hand, salt ions such as Mg2+, Al3+, ClO4-, SCN-, BF4-, are showed as "salting in" effect and water structure breakers by enhancing the hydration of copolymers, thus both CMC and cloud point increase [14,15]. Organic salts can also affect aggregation behaviors of copolymers, synergistic effects occur when organic salts are added to copolymer solutions [16,17]. It has been reported that there are hydrophobic and π-π interactions between sodium benzoate and alkylaryl groups in copolymer [18,19]. Organic salts differ from inorganic salts and differ from surfactants micellar solubilization for hydrophobic substances. It is not very explicit how organic salts affect the self-assemble behavior of copolymers, which should be further explored. Considering the extensive use of some pharmaceutical excipient salts, we systematically investigated the influence of three salts on the properties of two PEO-PPO block copolymers. Sodium chloride (NaCl) is a frequently used in parenteral formulations and in the production of isotonic solutions. Sodium citrate (NaCA) is an organic salt, which is commonly used as anticoagulant, expectorant and diuretic in pharmaceutical industries. Likewise, sodium benzoate (NaBZ) is an organic salt and served as preservatives in pharmacy. In this paper, the effects of NaCl, NaCA and NaBZ on micellization behavior of copolymers P123 and BP123 were investigated. BP123 was newly synthesized with two aromatic rings in the centre linked to PEO-PPO blocks. BP123 and P123 are a good pair of objects for comparing the impact of hydrophobic modification, as they have the same PEO and PPO segments. Moreover, the influence of NaCl, NaCA and NaBZ on drug simvastatin (SV) solubilization and drug release of P123 and BP123 were investigated. SV is used to lower bad cholesterol, triglycerides and the chance of heart attack. However, the poor water solubility of SV (only 1.8 ug·mL−1) impacted its bioavailability. The aim of this work is to determine how pharmaceutical excipient salts increase the solubility of SV and control the release of drug, providing reference for the application of copolymers as drug carrier for SV in pharmacy.
Copolymer solutions were added to 20 mL transparent tubes, then placed in a temperature-controlled shaker, and increased by 0.5℃ per minute. The temperature was recorded when the solution became turbidity, and the measurement was repeated 3 times. 2.4. Surface tension Surface tension of copolymer solutions at 25℃ was determined by an automatic JYW-200C surface tensiometer (the precise degree is 0.01 mN·m−1). The surface tension of distilled water was measured to calibrate the instrument. 2.5. Fluorescence Fluorescence was carried out by F-280 fluorescence spectrophotometer at 25℃. Pyrene was used as fluorescence probe with concentration of 1 × 10−6 mol·L-1. The excitation wavelength of pyrene spectrum was 335 nm, and the emission and excitation slits were set as 2.5 nm and 10 nm. 2.6. Dynamic light scattering Hydrodynamic diameter and size distribution of copolymer micelles were defined by Malvern Zetasizer Nano ZS. The scattering angle was 175°, using a He-Ne Laser. Each data ran three times at 25℃. 2.7. Drug solubilization Copolymer solution was added to each tube containing excessive drug. The tubes were shaken in the oscillator at 25 °C for 72 h. The undissolved drug was removed by cellulose acetate membrane. Then each sample was measured by TU-1901 UV–vis spectrophotometer, and drug concentration was calculated by the standard curve formula.
2. Materials and methods
2.8. In vitro drug release
2.1. Materials
Drug loaded copolymer solution (3 mL) was sealed in the dialysis bag, and then put in 200 mL of phosphate buffer (pH 7.4), with uninterruptedly stirred at 100 rpm and kept at 37 °C. At regular time lag, 2 mL of external medium was taken out and compensated by the buffer. Concentration of released drug was determined by UV–vis spectrophotometer.
Copolymer BP123 was synthesized in our laboratory. Pluronic copolymer P123 was a gift sample from BASF. Drug SV was provided by Wuhan Biocar BioPharm Co. The salts NaCl, NaCA, NaBZ and other reagents were purchased from Sinopharm Chemical Reagent Co. The general structures of copolymers, salts and drug are shown in Fig. 1(a).
3. Results and discussion 2.2. Synthesis and characterization of copolymers 3.1. Effect of salts on cloud point Copolymer BP123 was synthesized by anionic polymerization, according to our previous work [20,21]. BP123 and P123 were obtained by different initiators, the precursor of BP123 was bisphenol A and the precursor of P123 was propylene glycol. The precursor firstly reacted with propylene oxide and then with ethylene oxide. After obtaining the copolymer with designed molecular weight, the reaction was terminated. 1 H NMR analysis of copolymer was performed on a Bruker AV-400 NMR spectrometer. 1H NMR spectrum of BP123 in CDCl3 is displayed in Fig. 1(b). 1H NMR peaks at 6.7–7.4 ppm referred to the protons of aromatic rings. PPO/PEO ratio was obtained according to the peak at 1.0–1.1 ppm and 3.3–3.7 ppm. The former peaks were attributed to PPO−CH3, and the latter peaks were ascribed to PEO−CH2, PPO−CH2 and PPO−CH. NMR-based molecular weight (Mn) was calculated by comparing integrals of the side methyl of aromatic ring (Ar-C−CH3: 1.2 ppm) with the side methyl of PPO (PPO−CH3: 1.0–1.1 ppm). The validated PPO/PEO ratio and Mn of BP123 were in good agreement with the target amounts.
Cloud point is an important character exhibited by non-ionic surfactants and uncharged polymers. With the increase of temperature, aqueous solutions undergo phase separation and offer turbid appearance above cloud point [22]. Amphiphiles form large aggregates above cloud point, which could not get through the biomembrane and reduce pharmacological activity in drug delivery. Fig. 2 shows the influence of three salts on cloud point of 5 % BP123 and P123 aqueous solutions. For 5 % solution, a linear decrease of cloud point was observed with the increase of NaCl concentration. As reported, NaCl is a “salting out” electrolyte, which makes the cloud point decrease [23]. With the addition of NaCl, Na+ and Cl− ionized with water, and well hydrated salt ions drew back some water molecules from copolymer. The hydration of copolymer became weaken and the solubility of BP123 decreased. As a result, phase separation took place at a lower temperature. Cloud point of BP123 decreased with the increase of NaCA concentration. The effect of NaCA was similar to “salting-out” phenomenon 2
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Fig. 1. (a) Structures of copolymers, salts and drug; (b) 1H NMR spectrum of BP123 in CDCl3.
anion. Therefore, benzoate anion acted as “salting in” anion, which weakened the self-hydration and stimulated the hydration of copolymer, so the cloud point increased. The impact of three salts on cloud point of P123 is shown in Fig. 2(b). The variation rule was consistent with the result of BP123. However, the effect of salts on cloud point of BP123 was clearly greater than that of P123. This was because that when salts were added to copolymer solution, water surrounded the salt ions could be polarized by the ionic field [24]. Hydrophobic PPO chains and aromatic rings were connected in BP123 molecules, and aromatic rings in BP123 were more sensitive to the polarity of solution than P123, so the cloud point of BP123 had a significant change with salt addition.
of NaCl, and the impact of NaCA was more significant than NaCl. NaCA could be ionized to three Na+ and one citrate ion, and NaCl could be ionized to one Na+ and one Cl−. In order to maintain the same concentration of Na+, the concentration of NaCl is set three times as high as NaCA. In Fig. 2, the concentration gradient of NaCl was set as 0.15, 0.3, 0.45 and 0.6 mol·L-1, while the concentration gradient of NaCA was set as 0.05, 0.1, 0.15 and 0.2 mol·L-1, respectively. Therefore, the concentration of Na+ was the same for NaCl and NaCA, whereas the concentration of citrate ion in NaCA solution was much lower than that of Cl− in NaCl solution, which further illustrated that the influence of citrate ion was greater than that of Cl−. Citrate ion exhibited greater "salting out" effect than Cl−, and well hydrated citrate ions could strongly reduce the solubility of copolymer and the cloud point decreased significantly. Oppositely, cloud point of BP123 increased linearly with the increasing of NaBZ concentration. The concentration gradient of NaBZ was the same as that of NaCl. The effect of NaBZ was similar to “salting in” phenomenon of salts. NaBZ could be ionized to Na+ and benzoate
3.2. Effect of salts on surface tension Fig. 3 reveals the influence of salts on surface tension of BP123 and P123 solutions. Without salts, surface tension decreased with the increase of copolymer concentration for BP123 and P123, and there were
Fig. 2. Cloud point of (a) 5 % BP123 (b) 5 % P123 with different salts. 3
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Fig. 3. Surface tension of (a) BP123 (b) P123 with different salts.
two breaks in the curves. The first break was caused by the molecular conformational changes of copolymer at the air/water surface [25,26]. The second break meant that the adsorption of copolymer at the surface became saturated. Afterwards, a platform appeared in the curve, and copolymer began to aggregate in the solution, so the second break was the real CMC of copolymer. The concentration of three salts was chosen to maintain the same concentration of Na+, the concentration of NaCl and NaBZ was 0.3 mol·L−1, and the concentration of NaCA was 0.1 mol·L−1, which was the intermediate concentration in the cloud point experimental range. The addition of NaCl or NaCA transformed CMC to a lower concentration. The strong hydration of NaCl or NaCA could capture water molecules surrounding copolymer molecules, thus increasing the hydrophobicity of copolymer. The addition of NaCA had much greater influence than NaCl, which was consistent with their effects on cloud point. Since each NaCA molecule had several hydrogen donors and receptors, NaCA acted as a good water structure maker which interacted favorably with water and it could strongly affect the hydrogen bond network of solution, thus increasing hydrophobic interaction and decreasing CMC distinctly. The surface tension at CMC (γCMC) of pure BP123 was 33.34 mN·m−1. In the presence of NaCA or NaCl, surface tension decreased slightly within the whole copolymer concentration range, and γCMC values were 32.87 and 31.89 mN·m−1, respectively. The addition of NaCA or NaCl could enhance the dehydration of the PPO block and break the hydration layers of PEO groups. As a result, the adsorption of the copolymer on the surface showed more favorably, and the surface tension decreased. Furthermore, NaCl or NaCA acted as a water structure maker, which made the ice-like structure of water compact, resulting in a tighter packing of copolymer [27]. However, the effect of NaBZ on surface tension presented different characteristics, both CMC and γCMC increased. NaBZ manifested as "salting in" effect was proved by cloud point measurement, increasing the hydration of copolymer and reducing the hydrophobicity of
copolymer. Therefore, CMC of BP123 (0.05 %) increased to 0.1 % with the addition of NaBZ. Meanwhile, NaBZ could be considered as a water structure breaker, which made the ice-like structure of water loose and induced a lesser coiled of copolymer [28]. Thus, γCMC value increased. The surface tension of pure 0.3 mol·L−1 NaCl, 0.1 mol·L−1 NaCA and 0.3 mol·L−1 NaBZ is 72.85, 72.19 and 67.76 mN·m-1, respectively. The surface tension of pure water at 25℃ is 72.08 mN·m-1. Unlike NaCl and NaCA, NaBZ decreased surface tension, because NaBZ could adsorb on the air/water surface by hydrophobic aromatic ring. With further increase of BP123 concentration, BP123 molecules replaced some NaA molecules on the surface because the hydrophobicity of BP123 was stronger than NaA. Above CMC, NaBZ and BP123 molecules could coadsorb on the surface and form a mixed adsorbed layer, which generated γCMC of BP123 in the presence of NaA (34.46 mN·m-1) to be bigger than pure BP123 solution (33.34 mN·m-1). At the same time, NaBZ could be solubilized in BP123 micelles. In other words, BP123 and NaBZ could form mixed micelles, making CMC increase. As shown in Fig. 3(b), likewise for P123, the addition of NaCl or NaCA decreased CMC and γCMC, and the opposite effect was observed when NaBZ was added. It is worth noting that the distinction between pure BP123 and P123. Both CMC and γCMC of P123 were bigger than BP123. CMC of pure P123 was 0.2 %, and γCMC was 34.20 mN·m−1. CMC of pure BP123 was 0.1 %, and γCMC was 33.34 mN·m−1. Considering that PEO and PPO segments of P123 and BP123 were the same, aromatic rings in BP123 could enhance the hydrophobicity, thus decreasing CMC and raising the surface activity. 3.3. Effect of salts on pyrene fluorescence The spectrum of pyrene fluorescence in 350−420 nm consisted of five peaks, which are generally designated as I1-I5, from shorter to longer wavelengths. The ratio of I1/I3 intensity depended on microenvironment polarity. The decrease of I1/I3 ratio indicated encapsulation of pyrene into a hydrophobic environment, which provided
Fig. 4. Pyrene fluorescence I1/I3 ratio of (a) BP123 (b) P123 with different salts. 4
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behavior, resulting in more copolymers in each micelle structure. Nevertheless, in the presence of NaBZ, BP123 micelles presented a bimodal size distribution with a main fraction of 91.66 nm and a secondary size population at 12.51 nm. From one point of view, NaBZ could participate in the formation of micelles due to the aromatic ring of NaBZ, resulting in much bigger micellar size. From another point of view, NaBZ showed as "salting in" effect and water structure-breaker salt. NaBZ disfavored the assembly behavior, resulting in less copolymers in each micelle structure, and the micellar size (12.51 nm) was smaller than pure BP123 (16.86 nm). The primary size population was the larger micellar size, indicating that the formation of mixed micelles acted the main impact. The similar effect was observed for P123 with NaCl or NaCA as shown in Fig. 5(b), which induced the micellar size increased and presented a single population. The addition of NaBZ also had two kinds of influence factors, among the formation of mixed micelles was more important so the addition of NaBZ increased micellar size of P123, while it was not very prominent. With NaBZ, the micellar size just increased from 14.92 nm to 35.80 nm. P123 with NaBZ remained a single population, while the addition of NaBZ made BP123 transform to bimodal populations. The phenomenon further illustrated that there was strong influence by aromatic rings of BP123, which altered the micelle size distribution.
information about micellar interior [29]. I1/I3 values of pure BP123 and P123 solutions are shown in Fig. 4. At low copolymer concentration, I1/I3 ratio was quite high, showing an aqueous environment around pyrene. With the increase of copolymer concentration, I1/I3 dropped abruptly since short-lived oligomers started to form [30]. After the second inflection point, I1/I3 remained almost unchanged, proving the existence of steady micelles. The second inflection point was CMC, and after that a mass of pyrene molecules encapsulated into hydrophobic interior of micelles. The addition of NaCl or NaCA decreased the CMC of copolymer, while the addition of NaBZ increased CMC. It could also be seen that the CMC of pure BP123 (0.05 %) was lower than P123 (0.1 %), which were all consistent with the surface tension results. For BP123, I1/I3 value after the second inflection point was in the order of NaCA (1.38) < NaCl (1.39) < salt-free (1.40) < NaBZ (1.42). The decrease of I1/I3 value indicated the micellar core became more hydrophobic and compact. When NaCl or NaCA was added, micelles became more hydrophobic and compact due to the dehydration of PPO core by salts. NaBZ showed a different pattern of behavior. NaBZ greatly decreased I1/I3 value at low copolymer concentration, because NaBZ had hydrophobic aromatic ring, providing more hydrophobic environment around pyrene. Whereas, I1/I3 above CMC with NaBZ was higher than pure copolymer since NaBZ participated in the formation of micelles, which made the micelles loosen. It was also the reason why the addition of NaBZ increased CMC of copolymer, and they formed the mixed micelles at higher copolymer concentration. As shown in Fig. 4(b), the effect of salts on both CMC and I1/I3 of BP123 was more prominent than that of P123. This was consistent with the effect of cloud point. Aromatic rings in BP123 could interact with pyrene, which made less excimer form of pyrene in the systems [31]. Especially in the presence of NaBZ, there were strong interactions between benzoate anions and aromatic rings in BP123, and the effect was greater.
3.5. Effect of salts on drug solubilization The ability of copolymer micelles to host hydrophobic drugs and to serve as carriers was crucial for pharmaceutical applications [33]. Aqueous solubility of SV was only 1.8 ug·mL−1. In the absence of salts, SV solubility showed a dramatical increase to 218 μg mL−1 in 1 % BP123, and the data in 1 % P123 also climbed to 151 ug·mL−1, indicating that both BP123 and P123 micelles were able to solubilize a larger amount of SV. The higher solubility of BP123 compared with P123 could be ascribed to more hydrophobic nature of BP123. Solubilization of drug SV in BP123 and P123 solution as a function of salt concentration are shown in Fig. 6. The order of solubilization ability for BP123 and P123 was both NaBZ > NaCA > NaCl > saltfree. Micellar solubilization capacity was greatly influenced by the available volume of micelles for hydrophobic substances [34]. Micellar size in the presence of salts also followed the order NaBZ > NaCA > NaCl > salt-free, the addition of salts all increased size of micelles. Some other features were also related to solubilization ability of copolymers. NaCA and NaCl presented higher surface activity (Fig. 3), which wetted the surface of drug particles and assisted encapsulating drug in micelles. The higher solubilization of SV with NaCA or NaCl could also be attributed to the salting out effect of NaCA or NaCl, increasing the hydrophobicity of micelle cores (Fig. 4). The solubilization showed a remarkable increase when NaBZ was added to copolymer. On the one hand, it was ascribed to the much larger size of the mixed micelles provided to load drug. On the other
3.4. Effect of salts on micellar size Dynamic light scattering was frequently used to evaluate hydrodynamic size of micelles, which contained corona region of micelles with associated solvent molecules. So any changes in corona would be reflected in the measured size [32]. As shown in Fig. 5, the micellar size of pure BP123 (16.86 nm) was bigger than P123 (14.92 nm). Aromatic rings in BP123 could enhance the hydrophobicity, aggregation ability enhanced, which made micellar grow and more molecules form micelles. In Fig. 5(a), the addition of NaCl or NaCA induced micellar size increase from 16.86 nm to 26.25 nm and 43.82 nm. The effect of NaCA was stronger than NaCl because the “salting out” effect of citrate ion was greater than Cl−. They broke the hydration shell of micelles and raised the ability of hydrophobicity. NaCl and NaCA favored assembly
Fig. 5. Micellar size and polydispersity index values (PDI) of (a) 1 % BP123 (b) 1 % P123 with different salts. 5
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Fig. 6. Solubilization of (a) 1 % BP123 (b) 1 % P123 with different salts for drug SV.
added to BP123 or P123. The influence of NaBZ on drug release rate for P123 was not very notable, the order of release rate was NaCA < NaBZ < NaCl < salt-free. For BP123, the release rate was in the order of NaBZ < NaCA < NaCl < salt-free. Both NaBZ and BP123 had hydrophobic benzene rings, which greatly enhanced the interaction with hydrophobic drug, leading to markedly delay of the drug release in BP123. The probable mechanism of SV release from copolymer micelles were analyzed by mathematical models including zero-order, firstorder, and Higuchi equation models [36]. The rate constant (K) and correlation coeffificient (R2) were calculated for these three models and shown in Table 1. The criterion for selecting the most appropriate model was based on the largest goodness-of-fit (R2 values). The release of SV from pure 1 % BP123 and P123 micelles followed the first-order kinetics, the drug release rate depended on concentration, which was the dominant extended release pattern found in the pharmaceutical industry. Whereas, with the addition of different salts they followed Higuchi model, described that the drug release was controlled by diffusion.
hand, the existence of hydroxy and hydrophobic moieties in drug SV could interact with NaBZ. There were hydrophobic interactions, π-π interactions and hydrogen-bond interactions in the system, which further increased the solubilization of SV. 3.6. In vitro drug release To serve as drug carriers, it was crucial for micelles to entrap the drug for an extended period of time during circulation [35]. Fig. 7 shows the cumulative release profiles of drug SV versus time from BP123 and P123 solutions. The release rate of SV from copolymer solution was remarkable slower than that of pure SV, which represented the drug released from copolymer micelles was effectively retarded in a controlled and sustained way. Without salt, cumulative release amount reached 85 % and 97 % for pure BP123 and P123 respectively. The lower release rate of BP123 could be ascribed to the higher hydrophobicity of BP123 and stronger micelle/drug interaction, which could delay drug release rate. The addition of different salts remarkably affected the release rate of SV, which meant the release rate could be well managed by the addition of salts. NaCl and NaCA decelerated the release rate, since micelles became more compact and hydrophobic in the presence of NaCA and NaCl. Micelles with NaCA were more compact than that of NaCl, inducing a stronger micelle/drug interaction hence the drug release rate was much slower with NaCA. The addition of NaBZ also delayed the release rate. Although NaBZ increased I1/I3 value, it was supposed to release much faster due to the loose packing of micelles. However, another parameter managing the release process was the micelle/drug affinity. NaBZ participated in the formation of micelles, therefore the interaction between aromatic rings of drug and micelle was greater, which could hamper the release of drug. Additionally, a different performance occurred when NaBZ was
4. Conclusions The impact of NaCl, NaCA and NaBZ on self-assembly, drug solubilization and drug release property of hydrophobic modification PEOPPO copolymer BP123 and P123 were studied. NaCA and NaCl manifested "salting out" effect, reducing cloud point and CMC. The impact of NaCA was more significant than NaCl. They promoted the growth of micelles, enhanced drug solubilization and slowed the drug release rate. NaBZ was similar to “salting in” effect, both cloud point and CMC increased. The more crucial effect was that NaBZ participated in micelle formation duo to benzene ring, so micellar size and drug solubilization signally increased. Whereas, NaBZ displayed difference for BP123 and P123 drug release, because benzene rings in BP123 could strongly
Fig. 7. In vitro release profiles of free SV and SV-loaded different copolymers (a) 1 % BP123 (b) 1 % P123 with different salts at physiological conditions (37 °C, pH 7.4). 6
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Table 1 The release kinetic models depicting the release pattern of SV from copolymer micelles. Copolymer
1 % BP123 +0.3 mol·L−1 +0.1 mol·L−1 +0.3 mol·L−1 1 % P123 +0.3 mol·L−1 +0.1 mol·L−1 +0.3 mol·L−1
Zero-order model
NaCl NaCA NaBZ NaCl NaCA NaBZ
First-order model 2
K
R
1.1284 1.0719 1.0259 1.0435 1.3020 1.2369 1.1196 1.1932
0.6578 0.6812 0.7409 0.6930 0.6914 0.7228 0.7713 0.6884
interact with hydrophobic drug and NaBZ, leading to markedly delay release of drug for BP123. It reflected that both the addition of salts and the copolymer architecture changes could influence the performance. Widely used pharmaceutical excipient salts NaCl, NaCA and NaBZ can obviously effect micellization, drug solubilization and drug release property in different ways, which can be conveniently utilized in designing formulations. The copolymer BP123 with benzene ring exhibit better drug solubilization and drug controlled release property, which would be advantageous in drug delivery systems.
K
R
0.0636 0.0478 0.0326 0.0252 0.1474 0.1140 0.0423 0.0712
0.9033 0.8368 0.8002 0.8467 0.9891 0.9563 0.9175 0.9026
[11] [12] [13]
[14]
[15]
Declaration of Competing Interest [16]
The authors declare that there is no conflict of interest associated with this study.
[17]
CRediT authorship contribution statement Yaoyao Zhu: Writing - original draft. Mengran Chu: Methodology. Zixiao Wang: Methodology. Yutao Xue: Methodology. Bo Liu: Writing - review & editing. Jie Sun: Writing - review & editing. Teng Liu: Writing - review & editing, Supervision.
[18]
Acknowledgements
[20]
[19]
This work was supported by the National Natural Science Foundation of China (81903473); the Science and Technology Research Program of Shandong Academy of Medical Sciences (2017-54); and the Innovation Project of Shandong Academy of Medical Sciences.
[21] [22] [23]
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K
R2
8.9691 8.7277 8.3089 8.6050 10.600 10.070 8.7893 9.6199
0.8617 0.8817 0.8714 0.9132 0.8617 0.9617 0.9330 0.9224
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Pharmaceutical excipient salts effect on micellization and drug solubilization of PEO-PPO-ph-PPO-PEO block copolymer
T
Yaoyao Zhua,b, Mengran Chua,b, Zixiao Wanga,b, Yutao Xuea, Bo Liua, Jie Suna, Teng Liua,* a b
Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan 250062, PR China School of Medicine and Life Sciences, University of Jinan-Shandong Academy of Medical Sciences, Jinan, PR China
A R T I C LE I N FO
A B S T R A C T
Keywords: PEO-PPO copolymer Hydrophobic modification Sodium citrate Micelles Drug solubilization
Hydrophobic modification PEO-PPO copolymer BP123 was synthesized, with two aromatic rings in the centre linked to PEO-PPO blocks, and the identical PEO and PPO block numbers were possessed with commercial copolymer P123. The influence of three common pharmaceutical excipient salts sodium chloride (NaCl), sodium citrate (NaCA) and sodium benzoate (NaBZ) on self-assembly behaviors of BP123 and P123 was investigated via cloud point, surface tension, pyrene fluorescence and dynamic light scattering. Solubilization for hydrophobic drug simvastatin (SV) and in vitro drug release behavior were assessed accordingly. In the presence of NaCl or NaCA, cloud point and critical micellization concentration (CMC) decreased, micelles became more hydrophobic, micellar size and drug solubilization increased, drug release rate slowed, and the impact of NaCA was more significant than NaCl. Oppositely, cloud point and CMC increased with the addition of NaBZ. NaBZ could participate in the formation of micelles by hydrophobic aromatic ring, which greatly raised solubilization of SV. Moreover, a different performance occurred when NaBZ was added to BP123 or P123, due to the hydrophobic benzene rings in BP123, which prominently enhanced the interaction with hydrophobic drug, leading to obvious delay of drug release for BP123. This work is conducive to turning copolymer property in diverse pharmaceutical applications and in drug delivery systems as drug carriers.
1. Introduction Block copolymers comprise of poly (ethylene oxide) (PEO) and poly (propylene oxide) (PPO) blocks and are commercially available under the trade name Pluronic (PEO-PPO-PEO), which have been extensively studied from viewpoint of aqueous solution behaviors [1–3]. They selfassemble to form micelles in aqueous solutions, with PPO groups forming a fairly compact core and hydrated PEO groups constituting a swollen protective corona, and these core-shell micelles could effectively solubilize hydrophobic drugs. Pluronic copolymers are biocompatible, which have remarkable applications in controlled drug encapsulation systems since their micelles own a slower rate of dissociation and allow the preservation of loaded drugs for a longer period of time [4,5]. The aggregation behavior and solubilizing ability of Pluronic depend on various factors, such as copolymer structure, ionic strength and different additives. Chemical nature of copolymer obviously affects aggregation behavior and drug load ability. Molecular characteristics, such as PPO/PEO ratio and molecular weight, have been widely investigated [6,7]. When
Pluronic contains same PEO segments, critical micellization concentration (CMC) decreases as PPO content increases. Pluronic with higher PPO/PEO ratio possesses larger micelles and more hydrophobic cores, so its solubilization ability is better. Recently, many research groups have studied the influence of hydrophobic modification on the self-assemble process of copolymers [8,9]. The existence of hydrophobic groups in copolymer could notably raise the adsorption ability and strengthen aggregation behavior. Nevertheless, researches about hydrophobic modification of PEO-PPO copolymers are just rising and there remains much unknown information. Ionic strength clearly impacts the aggregation behavior of Pluronic. The classical order for common inorganic salt ions to influence the aggregation behaviors of copolymers is basically determined, according to Hofmeister series generally [10,11]. On the one hand, some salt ions are manifested as "salting out" effect and water structure makers, including K+, Na+, SO42−, Cl-, Br-, are characterized by weakening the hydration of copolymers as both CMC and cloud point decrease. Simultaneously, these ions can enhance the solubilization of hydrophobic drug, because they remove water from the core and improve the
⁎ Corresponding author at: Institute of Materia Medica, Shandong First Medical University & Shandong Academy of Medical Sciences, Jingshi Road No.18877, Jinan, 250062, PR China. E-mail address: [email protected] (T. Liu).
https://doi.org/10.1016/j.colsurfb.2020.110857 Received 19 October 2019; Received in revised form 14 January 2020; Accepted 10 February 2020 Available online 10 February 2020 0927-7765/ © 2020 Elsevier B.V. All rights reserved.
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2.3. Cloud point
hydrophobicity, as well as make more room to load drug [12,13]. On the other hand, salt ions such as Mg2+, Al3+, ClO4-, SCN-, BF4-, are showed as "salting in" effect and water structure breakers by enhancing the hydration of copolymers, thus both CMC and cloud point increase [14,15]. Organic salts can also affect aggregation behaviors of copolymers, synergistic effects occur when organic salts are added to copolymer solutions [16,17]. It has been reported that there are hydrophobic and π-π interactions between sodium benzoate and alkylaryl groups in copolymer [18,19]. Organic salts differ from inorganic salts and differ from surfactants micellar solubilization for hydrophobic substances. It is not very explicit how organic salts affect the self-assemble behavior of copolymers, which should be further explored. Considering the extensive use of some pharmaceutical excipient salts, we systematically investigated the influence of three salts on the properties of two PEO-PPO block copolymers. Sodium chloride (NaCl) is a frequently used in parenteral formulations and in the production of isotonic solutions. Sodium citrate (NaCA) is an organic salt, which is commonly used as anticoagulant, expectorant and diuretic in pharmaceutical industries. Likewise, sodium benzoate (NaBZ) is an organic salt and served as preservatives in pharmacy. In this paper, the effects of NaCl, NaCA and NaBZ on micellization behavior of copolymers P123 and BP123 were investigated. BP123 was newly synthesized with two aromatic rings in the centre linked to PEO-PPO blocks. BP123 and P123 are a good pair of objects for comparing the impact of hydrophobic modification, as they have the same PEO and PPO segments. Moreover, the influence of NaCl, NaCA and NaBZ on drug simvastatin (SV) solubilization and drug release of P123 and BP123 were investigated. SV is used to lower bad cholesterol, triglycerides and the chance of heart attack. However, the poor water solubility of SV (only 1.8 ug·mL−1) impacted its bioavailability. The aim of this work is to determine how pharmaceutical excipient salts increase the solubility of SV and control the release of drug, providing reference for the application of copolymers as drug carrier for SV in pharmacy.
Copolymer solutions were added to 20 mL transparent tubes, then placed in a temperature-controlled shaker, and increased by 0.5℃ per minute. The temperature was recorded when the solution became turbidity, and the measurement was repeated 3 times. 2.4. Surface tension Surface tension of copolymer solutions at 25℃ was determined by an automatic JYW-200C surface tensiometer (the precise degree is 0.01 mN·m−1). The surface tension of distilled water was measured to calibrate the instrument. 2.5. Fluorescence Fluorescence was carried out by F-280 fluorescence spectrophotometer at 25℃. Pyrene was used as fluorescence probe with concentration of 1 × 10−6 mol·L-1. The excitation wavelength of pyrene spectrum was 335 nm, and the emission and excitation slits were set as 2.5 nm and 10 nm. 2.6. Dynamic light scattering Hydrodynamic diameter and size distribution of copolymer micelles were defined by Malvern Zetasizer Nano ZS. The scattering angle was 175°, using a He-Ne Laser. Each data ran three times at 25℃. 2.7. Drug solubilization Copolymer solution was added to each tube containing excessive drug. The tubes were shaken in the oscillator at 25 °C for 72 h. The undissolved drug was removed by cellulose acetate membrane. Then each sample was measured by TU-1901 UV–vis spectrophotometer, and drug concentration was calculated by the standard curve formula.
2. Materials and methods
2.8. In vitro drug release
2.1. Materials
Drug loaded copolymer solution (3 mL) was sealed in the dialysis bag, and then put in 200 mL of phosphate buffer (pH 7.4), with uninterruptedly stirred at 100 rpm and kept at 37 °C. At regular time lag, 2 mL of external medium was taken out and compensated by the buffer. Concentration of released drug was determined by UV–vis spectrophotometer.
Copolymer BP123 was synthesized in our laboratory. Pluronic copolymer P123 was a gift sample from BASF. Drug SV was provided by Wuhan Biocar BioPharm Co. The salts NaCl, NaCA, NaBZ and other reagents were purchased from Sinopharm Chemical Reagent Co. The general structures of copolymers, salts and drug are shown in Fig. 1(a).
3. Results and discussion 2.2. Synthesis and characterization of copolymers 3.1. Effect of salts on cloud point Copolymer BP123 was synthesized by anionic polymerization, according to our previous work [20,21]. BP123 and P123 were obtained by different initiators, the precursor of BP123 was bisphenol A and the precursor of P123 was propylene glycol. The precursor firstly reacted with propylene oxide and then with ethylene oxide. After obtaining the copolymer with designed molecular weight, the reaction was terminated. 1 H NMR analysis of copolymer was performed on a Bruker AV-400 NMR spectrometer. 1H NMR spectrum of BP123 in CDCl3 is displayed in Fig. 1(b). 1H NMR peaks at 6.7–7.4 ppm referred to the protons of aromatic rings. PPO/PEO ratio was obtained according to the peak at 1.0–1.1 ppm and 3.3–3.7 ppm. The former peaks were attributed to PPO−CH3, and the latter peaks were ascribed to PEO−CH2, PPO−CH2 and PPO−CH. NMR-based molecular weight (Mn) was calculated by comparing integrals of the side methyl of aromatic ring (Ar-C−CH3: 1.2 ppm) with the side methyl of PPO (PPO−CH3: 1.0–1.1 ppm). The validated PPO/PEO ratio and Mn of BP123 were in good agreement with the target amounts.
Cloud point is an important character exhibited by non-ionic surfactants and uncharged polymers. With the increase of temperature, aqueous solutions undergo phase separation and offer turbid appearance above cloud point [22]. Amphiphiles form large aggregates above cloud point, which could not get through the biomembrane and reduce pharmacological activity in drug delivery. Fig. 2 shows the influence of three salts on cloud point of 5 % BP123 and P123 aqueous solutions. For 5 % solution, a linear decrease of cloud point was observed with the increase of NaCl concentration. As reported, NaCl is a “salting out” electrolyte, which makes the cloud point decrease [23]. With the addition of NaCl, Na+ and Cl− ionized with water, and well hydrated salt ions drew back some water molecules from copolymer. The hydration of copolymer became weaken and the solubility of BP123 decreased. As a result, phase separation took place at a lower temperature. Cloud point of BP123 decreased with the increase of NaCA concentration. The effect of NaCA was similar to “salting-out” phenomenon 2
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Fig. 1. (a) Structures of copolymers, salts and drug; (b) 1H NMR spectrum of BP123 in CDCl3.
anion. Therefore, benzoate anion acted as “salting in” anion, which weakened the self-hydration and stimulated the hydration of copolymer, so the cloud point increased. The impact of three salts on cloud point of P123 is shown in Fig. 2(b). The variation rule was consistent with the result of BP123. However, the effect of salts on cloud point of BP123 was clearly greater than that of P123. This was because that when salts were added to copolymer solution, water surrounded the salt ions could be polarized by the ionic field [24]. Hydrophobic PPO chains and aromatic rings were connected in BP123 molecules, and aromatic rings in BP123 were more sensitive to the polarity of solution than P123, so the cloud point of BP123 had a significant change with salt addition.
of NaCl, and the impact of NaCA was more significant than NaCl. NaCA could be ionized to three Na+ and one citrate ion, and NaCl could be ionized to one Na+ and one Cl−. In order to maintain the same concentration of Na+, the concentration of NaCl is set three times as high as NaCA. In Fig. 2, the concentration gradient of NaCl was set as 0.15, 0.3, 0.45 and 0.6 mol·L-1, while the concentration gradient of NaCA was set as 0.05, 0.1, 0.15 and 0.2 mol·L-1, respectively. Therefore, the concentration of Na+ was the same for NaCl and NaCA, whereas the concentration of citrate ion in NaCA solution was much lower than that of Cl− in NaCl solution, which further illustrated that the influence of citrate ion was greater than that of Cl−. Citrate ion exhibited greater "salting out" effect than Cl−, and well hydrated citrate ions could strongly reduce the solubility of copolymer and the cloud point decreased significantly. Oppositely, cloud point of BP123 increased linearly with the increasing of NaBZ concentration. The concentration gradient of NaBZ was the same as that of NaCl. The effect of NaBZ was similar to “salting in” phenomenon of salts. NaBZ could be ionized to Na+ and benzoate
3.2. Effect of salts on surface tension Fig. 3 reveals the influence of salts on surface tension of BP123 and P123 solutions. Without salts, surface tension decreased with the increase of copolymer concentration for BP123 and P123, and there were
Fig. 2. Cloud point of (a) 5 % BP123 (b) 5 % P123 with different salts. 3
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Fig. 3. Surface tension of (a) BP123 (b) P123 with different salts.
two breaks in the curves. The first break was caused by the molecular conformational changes of copolymer at the air/water surface [25,26]. The second break meant that the adsorption of copolymer at the surface became saturated. Afterwards, a platform appeared in the curve, and copolymer began to aggregate in the solution, so the second break was the real CMC of copolymer. The concentration of three salts was chosen to maintain the same concentration of Na+, the concentration of NaCl and NaBZ was 0.3 mol·L−1, and the concentration of NaCA was 0.1 mol·L−1, which was the intermediate concentration in the cloud point experimental range. The addition of NaCl or NaCA transformed CMC to a lower concentration. The strong hydration of NaCl or NaCA could capture water molecules surrounding copolymer molecules, thus increasing the hydrophobicity of copolymer. The addition of NaCA had much greater influence than NaCl, which was consistent with their effects on cloud point. Since each NaCA molecule had several hydrogen donors and receptors, NaCA acted as a good water structure maker which interacted favorably with water and it could strongly affect the hydrogen bond network of solution, thus increasing hydrophobic interaction and decreasing CMC distinctly. The surface tension at CMC (γCMC) of pure BP123 was 33.34 mN·m−1. In the presence of NaCA or NaCl, surface tension decreased slightly within the whole copolymer concentration range, and γCMC values were 32.87 and 31.89 mN·m−1, respectively. The addition of NaCA or NaCl could enhance the dehydration of the PPO block and break the hydration layers of PEO groups. As a result, the adsorption of the copolymer on the surface showed more favorably, and the surface tension decreased. Furthermore, NaCl or NaCA acted as a water structure maker, which made the ice-like structure of water compact, resulting in a tighter packing of copolymer [27]. However, the effect of NaBZ on surface tension presented different characteristics, both CMC and γCMC increased. NaBZ manifested as "salting in" effect was proved by cloud point measurement, increasing the hydration of copolymer and reducing the hydrophobicity of
copolymer. Therefore, CMC of BP123 (0.05 %) increased to 0.1 % with the addition of NaBZ. Meanwhile, NaBZ could be considered as a water structure breaker, which made the ice-like structure of water loose and induced a lesser coiled of copolymer [28]. Thus, γCMC value increased. The surface tension of pure 0.3 mol·L−1 NaCl, 0.1 mol·L−1 NaCA and 0.3 mol·L−1 NaBZ is 72.85, 72.19 and 67.76 mN·m-1, respectively. The surface tension of pure water at 25℃ is 72.08 mN·m-1. Unlike NaCl and NaCA, NaBZ decreased surface tension, because NaBZ could adsorb on the air/water surface by hydrophobic aromatic ring. With further increase of BP123 concentration, BP123 molecules replaced some NaA molecules on the surface because the hydrophobicity of BP123 was stronger than NaA. Above CMC, NaBZ and BP123 molecules could coadsorb on the surface and form a mixed adsorbed layer, which generated γCMC of BP123 in the presence of NaA (34.46 mN·m-1) to be bigger than pure BP123 solution (33.34 mN·m-1). At the same time, NaBZ could be solubilized in BP123 micelles. In other words, BP123 and NaBZ could form mixed micelles, making CMC increase. As shown in Fig. 3(b), likewise for P123, the addition of NaCl or NaCA decreased CMC and γCMC, and the opposite effect was observed when NaBZ was added. It is worth noting that the distinction between pure BP123 and P123. Both CMC and γCMC of P123 were bigger than BP123. CMC of pure P123 was 0.2 %, and γCMC was 34.20 mN·m−1. CMC of pure BP123 was 0.1 %, and γCMC was 33.34 mN·m−1. Considering that PEO and PPO segments of P123 and BP123 were the same, aromatic rings in BP123 could enhance the hydrophobicity, thus decreasing CMC and raising the surface activity. 3.3. Effect of salts on pyrene fluorescence The spectrum of pyrene fluorescence in 350−420 nm consisted of five peaks, which are generally designated as I1-I5, from shorter to longer wavelengths. The ratio of I1/I3 intensity depended on microenvironment polarity. The decrease of I1/I3 ratio indicated encapsulation of pyrene into a hydrophobic environment, which provided
Fig. 4. Pyrene fluorescence I1/I3 ratio of (a) BP123 (b) P123 with different salts. 4
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behavior, resulting in more copolymers in each micelle structure. Nevertheless, in the presence of NaBZ, BP123 micelles presented a bimodal size distribution with a main fraction of 91.66 nm and a secondary size population at 12.51 nm. From one point of view, NaBZ could participate in the formation of micelles due to the aromatic ring of NaBZ, resulting in much bigger micellar size. From another point of view, NaBZ showed as "salting in" effect and water structure-breaker salt. NaBZ disfavored the assembly behavior, resulting in less copolymers in each micelle structure, and the micellar size (12.51 nm) was smaller than pure BP123 (16.86 nm). The primary size population was the larger micellar size, indicating that the formation of mixed micelles acted the main impact. The similar effect was observed for P123 with NaCl or NaCA as shown in Fig. 5(b), which induced the micellar size increased and presented a single population. The addition of NaBZ also had two kinds of influence factors, among the formation of mixed micelles was more important so the addition of NaBZ increased micellar size of P123, while it was not very prominent. With NaBZ, the micellar size just increased from 14.92 nm to 35.80 nm. P123 with NaBZ remained a single population, while the addition of NaBZ made BP123 transform to bimodal populations. The phenomenon further illustrated that there was strong influence by aromatic rings of BP123, which altered the micelle size distribution.
information about micellar interior [29]. I1/I3 values of pure BP123 and P123 solutions are shown in Fig. 4. At low copolymer concentration, I1/I3 ratio was quite high, showing an aqueous environment around pyrene. With the increase of copolymer concentration, I1/I3 dropped abruptly since short-lived oligomers started to form [30]. After the second inflection point, I1/I3 remained almost unchanged, proving the existence of steady micelles. The second inflection point was CMC, and after that a mass of pyrene molecules encapsulated into hydrophobic interior of micelles. The addition of NaCl or NaCA decreased the CMC of copolymer, while the addition of NaBZ increased CMC. It could also be seen that the CMC of pure BP123 (0.05 %) was lower than P123 (0.1 %), which were all consistent with the surface tension results. For BP123, I1/I3 value after the second inflection point was in the order of NaCA (1.38) < NaCl (1.39) < salt-free (1.40) < NaBZ (1.42). The decrease of I1/I3 value indicated the micellar core became more hydrophobic and compact. When NaCl or NaCA was added, micelles became more hydrophobic and compact due to the dehydration of PPO core by salts. NaBZ showed a different pattern of behavior. NaBZ greatly decreased I1/I3 value at low copolymer concentration, because NaBZ had hydrophobic aromatic ring, providing more hydrophobic environment around pyrene. Whereas, I1/I3 above CMC with NaBZ was higher than pure copolymer since NaBZ participated in the formation of micelles, which made the micelles loosen. It was also the reason why the addition of NaBZ increased CMC of copolymer, and they formed the mixed micelles at higher copolymer concentration. As shown in Fig. 4(b), the effect of salts on both CMC and I1/I3 of BP123 was more prominent than that of P123. This was consistent with the effect of cloud point. Aromatic rings in BP123 could interact with pyrene, which made less excimer form of pyrene in the systems [31]. Especially in the presence of NaBZ, there were strong interactions between benzoate anions and aromatic rings in BP123, and the effect was greater.
3.5. Effect of salts on drug solubilization The ability of copolymer micelles to host hydrophobic drugs and to serve as carriers was crucial for pharmaceutical applications [33]. Aqueous solubility of SV was only 1.8 ug·mL−1. In the absence of salts, SV solubility showed a dramatical increase to 218 μg mL−1 in 1 % BP123, and the data in 1 % P123 also climbed to 151 ug·mL−1, indicating that both BP123 and P123 micelles were able to solubilize a larger amount of SV. The higher solubility of BP123 compared with P123 could be ascribed to more hydrophobic nature of BP123. Solubilization of drug SV in BP123 and P123 solution as a function of salt concentration are shown in Fig. 6. The order of solubilization ability for BP123 and P123 was both NaBZ > NaCA > NaCl > saltfree. Micellar solubilization capacity was greatly influenced by the available volume of micelles for hydrophobic substances [34]. Micellar size in the presence of salts also followed the order NaBZ > NaCA > NaCl > salt-free, the addition of salts all increased size of micelles. Some other features were also related to solubilization ability of copolymers. NaCA and NaCl presented higher surface activity (Fig. 3), which wetted the surface of drug particles and assisted encapsulating drug in micelles. The higher solubilization of SV with NaCA or NaCl could also be attributed to the salting out effect of NaCA or NaCl, increasing the hydrophobicity of micelle cores (Fig. 4). The solubilization showed a remarkable increase when NaBZ was added to copolymer. On the one hand, it was ascribed to the much larger size of the mixed micelles provided to load drug. On the other
3.4. Effect of salts on micellar size Dynamic light scattering was frequently used to evaluate hydrodynamic size of micelles, which contained corona region of micelles with associated solvent molecules. So any changes in corona would be reflected in the measured size [32]. As shown in Fig. 5, the micellar size of pure BP123 (16.86 nm) was bigger than P123 (14.92 nm). Aromatic rings in BP123 could enhance the hydrophobicity, aggregation ability enhanced, which made micellar grow and more molecules form micelles. In Fig. 5(a), the addition of NaCl or NaCA induced micellar size increase from 16.86 nm to 26.25 nm and 43.82 nm. The effect of NaCA was stronger than NaCl because the “salting out” effect of citrate ion was greater than Cl−. They broke the hydration shell of micelles and raised the ability of hydrophobicity. NaCl and NaCA favored assembly
Fig. 5. Micellar size and polydispersity index values (PDI) of (a) 1 % BP123 (b) 1 % P123 with different salts. 5
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Fig. 6. Solubilization of (a) 1 % BP123 (b) 1 % P123 with different salts for drug SV.
added to BP123 or P123. The influence of NaBZ on drug release rate for P123 was not very notable, the order of release rate was NaCA < NaBZ < NaCl < salt-free. For BP123, the release rate was in the order of NaBZ < NaCA < NaCl < salt-free. Both NaBZ and BP123 had hydrophobic benzene rings, which greatly enhanced the interaction with hydrophobic drug, leading to markedly delay of the drug release in BP123. The probable mechanism of SV release from copolymer micelles were analyzed by mathematical models including zero-order, firstorder, and Higuchi equation models [36]. The rate constant (K) and correlation coeffificient (R2) were calculated for these three models and shown in Table 1. The criterion for selecting the most appropriate model was based on the largest goodness-of-fit (R2 values). The release of SV from pure 1 % BP123 and P123 micelles followed the first-order kinetics, the drug release rate depended on concentration, which was the dominant extended release pattern found in the pharmaceutical industry. Whereas, with the addition of different salts they followed Higuchi model, described that the drug release was controlled by diffusion.
hand, the existence of hydroxy and hydrophobic moieties in drug SV could interact with NaBZ. There were hydrophobic interactions, π-π interactions and hydrogen-bond interactions in the system, which further increased the solubilization of SV. 3.6. In vitro drug release To serve as drug carriers, it was crucial for micelles to entrap the drug for an extended period of time during circulation [35]. Fig. 7 shows the cumulative release profiles of drug SV versus time from BP123 and P123 solutions. The release rate of SV from copolymer solution was remarkable slower than that of pure SV, which represented the drug released from copolymer micelles was effectively retarded in a controlled and sustained way. Without salt, cumulative release amount reached 85 % and 97 % for pure BP123 and P123 respectively. The lower release rate of BP123 could be ascribed to the higher hydrophobicity of BP123 and stronger micelle/drug interaction, which could delay drug release rate. The addition of different salts remarkably affected the release rate of SV, which meant the release rate could be well managed by the addition of salts. NaCl and NaCA decelerated the release rate, since micelles became more compact and hydrophobic in the presence of NaCA and NaCl. Micelles with NaCA were more compact than that of NaCl, inducing a stronger micelle/drug interaction hence the drug release rate was much slower with NaCA. The addition of NaBZ also delayed the release rate. Although NaBZ increased I1/I3 value, it was supposed to release much faster due to the loose packing of micelles. However, another parameter managing the release process was the micelle/drug affinity. NaBZ participated in the formation of micelles, therefore the interaction between aromatic rings of drug and micelle was greater, which could hamper the release of drug. Additionally, a different performance occurred when NaBZ was
4. Conclusions The impact of NaCl, NaCA and NaBZ on self-assembly, drug solubilization and drug release property of hydrophobic modification PEOPPO copolymer BP123 and P123 were studied. NaCA and NaCl manifested "salting out" effect, reducing cloud point and CMC. The impact of NaCA was more significant than NaCl. They promoted the growth of micelles, enhanced drug solubilization and slowed the drug release rate. NaBZ was similar to “salting in” effect, both cloud point and CMC increased. The more crucial effect was that NaBZ participated in micelle formation duo to benzene ring, so micellar size and drug solubilization signally increased. Whereas, NaBZ displayed difference for BP123 and P123 drug release, because benzene rings in BP123 could strongly
Fig. 7. In vitro release profiles of free SV and SV-loaded different copolymers (a) 1 % BP123 (b) 1 % P123 with different salts at physiological conditions (37 °C, pH 7.4). 6
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Table 1 The release kinetic models depicting the release pattern of SV from copolymer micelles. Copolymer
1 % BP123 +0.3 mol·L−1 +0.1 mol·L−1 +0.3 mol·L−1 1 % P123 +0.3 mol·L−1 +0.1 mol·L−1 +0.3 mol·L−1
Zero-order model
NaCl NaCA NaBZ NaCl NaCA NaBZ
First-order model 2
K
R
1.1284 1.0719 1.0259 1.0435 1.3020 1.2369 1.1196 1.1932
0.6578 0.6812 0.7409 0.6930 0.6914 0.7228 0.7713 0.6884
interact with hydrophobic drug and NaBZ, leading to markedly delay release of drug for BP123. It reflected that both the addition of salts and the copolymer architecture changes could influence the performance. Widely used pharmaceutical excipient salts NaCl, NaCA and NaBZ can obviously effect micellization, drug solubilization and drug release property in different ways, which can be conveniently utilized in designing formulations. The copolymer BP123 with benzene ring exhibit better drug solubilization and drug controlled release property, which would be advantageous in drug delivery systems.
K
R
0.0636 0.0478 0.0326 0.0252 0.1474 0.1140 0.0423 0.0712
0.9033 0.8368 0.8002 0.8467 0.9891 0.9563 0.9175 0.9026
[11] [12] [13]
[14]
[15]
Declaration of Competing Interest [16]
The authors declare that there is no conflict of interest associated with this study.
[17]
CRediT authorship contribution statement Yaoyao Zhu: Writing - original draft. Mengran Chu: Methodology. Zixiao Wang: Methodology. Yutao Xue: Methodology. Bo Liu: Writing - review & editing. Jie Sun: Writing - review & editing. Teng Liu: Writing - review & editing, Supervision.
[18]
Acknowledgements
[20]
[19]
This work was supported by the National Natural Science Foundation of China (81903473); the Science and Technology Research Program of Shandong Academy of Medical Sciences (2017-54); and the Innovation Project of Shandong Academy of Medical Sciences.
[21] [22] [23]
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