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Novel self-assembled pH-responsive biomimetic nanocarriers for drug delivery
Materials Science and Engineering C 64 (2016) 346–353
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Novel self-assembled pH-responsive biomimetic nanocarriers for drug delivery Minming Wu, Zhaoyu Cao, Yunfei Zhao, Rong Zeng ⁎, Mei Tu, Jianhao Zhao Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, PR China
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 8 March 2016 Accepted 29 March 2016 Available online 4 April 2016 Keywords: Biomimetic pH-responsive Chitosan Nanocarriers Controlled release
a b s t r a c t Novel pH-responsive biodegradable biomimetic nanocarriers were prepared by the self-assembly of N-acetyl-Lhistidine-phosphorylcholine-chitosan conjugate (NAcHis-PCCs), which was synthesized via Atherton-Todd reaction to couple biomembrane-like phosphorylcholine (PC) groups, and N,N′-carbonyldiimidazole (CDI) coupling reaction to link pH-responsive N-acetyl-L-histidine (NAcHis) moieties to chitosan. In vitro biological assay revealed that NAcHis-PCCs nanoparticles had excellent biocompatibility to avoid adverse biological response mainly owing to their biomimetic PC shell, and DLS results confirmed their pH-responsive behavior in acidic aqueous solution (pH ≤ 6.0). Quercetin (QUE), an anti-inflammatory, antioxidant and potential anti-tumor hydrophobic drug, was effectively loaded in NAcHis-PCCs nanocarriers and showed a pH-triggered release behavior with the enhanced QUE release at acidic pH 5.5 compared to neutral pH 7.4. The results indicated that pHresponsive biomimetic NAcHis-PCCs nanocarriers might have great potential for site-specific delivery to pathological acidic microenvironment avoiding unfavorable biological response. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Stimuli-responsive polymeric nanocarriers have attracted great attention for the controlled delivery of bioactive agents like proteins, oligonucleotides, or small-molecular drugs to specific sites in vivo to enhance therapy efficacy, which can deliver and release the cargo to specific biological environment due to their abrupt physical and chemical changes triggered by specific chemical, biochemical, or physical stimuli, such as pH, enzyme, light and temperature [1,2]. In particularly, pH-responsive nanocarriers have been extensively developed for passive targeting to some pathological sites with metabolic acidic microenvironment relative to normal physiological pH (i.e., 7.4), such as solid tumors, chronic obstructive pulmonary disease (COPD), neuromuscular diseases and rheumatoid arthritis [3]; as well as improving the intracellular delivery efficacy of therapeutic/diagnostic agents due to the intracellular pH gradients (pH 5.9–6.2 in early endosomes, pH 5.0–5.5 in late endosomes and lysosomes, and neutral in cytosol) [4]. In the past decades, a number of pH-responsive polymeric nanocarriers have been successfully fabricated based on the incorporation of the moieties with pH-tunable hydrophilic/hydrophobicity including carboxyl and/or tertiary amino groups, or the pH-cleavable linkages such as hydrazone, hydrazide and acetal [2]. Lee et al. reported a pH-sensitive poly (tetrahydropyran-2-yl methacrylate) nanoparticles for local drug delivery to acidic inflammatory environments [5]. Park et al. reported pHsensitive N-acetyl histidine-conjugated glycol chitosan self-assembled ⁎ Corresponding author. E-mail address: [email protected] (R. Zeng).
http://dx.doi.org/10.1016/j.msec.2016.03.099 0928-4931/© 2016 Elsevier B.V. All rights reserved.
nanoparticles for intracytoplasmic delivery of the anticancer drug paclitaxel [6]. However, it's still a challenge to avoid unfavorable biological response in vivo, e.g. the rapid clearance by the natural defence mechanisms of the body, before they made the response triggered by acidic pH. Recently, cell membrane mimic phosphorylcholine (PC) has received significant interest to modify nanocarriers for avoiding the undesired biological response instead of the traditional poly(ethylene glycol) functionalization, since recent papers reported some unfavorable biological responses related to PEGylated nanocarriers, including accelerated blood clearance (ABC) phenomenon, hypersensitivity and inhibiting cellular uptake [7], while PC has excellent hemocompatibility and protein-resistant property mainly due to its strong hydration capacity via electrostatic interactions [8], and PC-based nanosystems could enter living cells through fusogenic interaction with the plasma membrane [9]. Zhao et al. reported that polymer micelles bearing cell outer membrane phosphorylcholine can minimize reticuloendothelial system clearance and circulate for long time to reach the target [10]. In our previous work, we have successfully constructed bio-inspired nanocarriers with excellent biocompatibility based on the self-assembly of biodegradable amphiphilic biomimetic deoxycholic acid (DCA)-PC-chitosan derivative for hydrophobic drug or protein delivery [11,12]. Quercetin (QUE), a well-known anti-inflammatory, antioxidant and potential anti-tumor flavonoid drug, has broad employments in biological, pharmacological and medical benefits [13,14]. It has been found that QUE played important roles in several anti-cancer therapies, such as treatment of human breast cancer, lung cancer, and cervical cancer in ways of antioxidative activities, inhibition of cell circle proliferation
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and enzymes activities [15–17]. In spite of the numerous advantages QUE bears, the clinical application of QUE was restrained by its poor water-solubility and bioavailability. In this work, we reported a novel approach to construct pH-responsive biomimetic nanocarriers for sitespecific delivery of QUE to tumor or inflammatory acidic microenvironment based on the self-assembly of biodegradable biomimetic chitosan derivative, N-acetyl-L-histidine-phosphorylcholine-chitosan (NAcHisPCCs), which possessed hydrophilic PC groups to avoid the undesired biological response caused by non-specific protein adsorption, and Nacetyl-L-histidine (NAcHis) groups as pH-responsive moieties, since the pKa of the imidazole group in histidine is around 6.5, which would be protonated to be hydrophilic to destabilize nanocarriers and eventually release cargo when milieu pH was below 6.5 [6]. The physicochemical properties, in vitro biocompatibility and pH-dependent drug release behavior of nanocarriers were investigated.
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2.3. Preparation of NAcHis-PCCs and QUE/NAcHis-PCCs nanoparticles Blank NAcHis-PCCs and QUE/NAcHis-PCCs nanoparticles were prepared by a solvent evaporation method [20]. Briefly, NAcHis-PCCs (10 mg) was dissolved in H2O (1 mL), diluted with ethanol (2 mL), and mixed with 1 mg QUE or not, and then rotate evaporated at 40 °C to obtain a thin film. After addition of 10 mL H2O, the solution was gently stirred for 5 min, filtered through a 0.45 μm Millipore filter and lyophilized to obtain the nanoparticles. The QUE loading efficiency (LE, amount of QUE loaded in nanoparticles/total amount of QUE in added solution) and loading capacity (LC, amount of QUE loaded in nanoparticles/mass of QUE-loaded nanoparticles) of NAcHis-PCCs nanoparticles were evaluated using UPLC (Acquity UPLC Waters, US, detection wavelength: 374 nm, BEH-Phenyl column, Eluent: 0.1% formic acid in water: methanol (40:60 v/v), Flow rate: 0.4 mL/min) as described in our previous work [11].
2. Experimental 2.1. Materials Chitosan (Mw = 29 k, Deacetylation degree: 100%) was obtained by repeating N-deacetylation of chitosan (Brookfield viscosity: 20 cps, Sigma-Aldrich). Diphenyl phosphite, pyrene and choline chloride was obtained from Acros Organics. L-PEI (Mw = 25 k) was purchased from Alfa-Aesar. N-acetyl-L-histidine monohydrate (NAcHis), N,N ′-carbonyldiimidazole (CDI) and quercetin (QUE) were purchased from Aladdin Reagent Co., Ltd. Other chemicals and solvents were of analytical grade. 2.2. Synthesis of NAcHis-PCCs All experiments involving water-sensitive compounds were conducted under dry conditions. Firstly, PCCs was synthesized according to our previous work [18]. Chitosan with deacetylation degree of 100% was obtained by treatment with 40% (w/v) NaOH aqueous solution at 110 °C for 1.5 h three times, and confirmed by 1H NMR, then modified to 6-O-trityl chitosan according to Nishimura's method [19]. 200 mg (0.5 mmol of free NH2) 6-O-trityl chitosan was dissolved in a mixed solution of dimethylacetamide (DMA, 10.0 mL), triethylamine (1.05 mL, 7.5 mmol) and tetrachloromethane (0.475 mL, 5.0 mmol) (Solution A). 740 mg (5.3 mmol) of choline chloride was dissolved in 20.0 mL of freshly distilled pyridine/DMSO (1:10) at room temperature, then 0.475 mL (2.5 mmol) of diphenyl phosphite was added and stirred for 2 h. After evaporation, the crude product of dicholinyl H-phosphonate dichloride was dissolved in 15 mL of isopropanol (Solution B). Solution B was added dropwise to solution A in ice-water bath, stirred for 12 h, then the resulting solution was evaporated to dryness. 15 mL of formic acid was added and stirred for 1 h under room temperature, then rotary evaporation was used to remove the remaining formic acid. The residue was dissolved in distilled water (20 mL) and filtered, supernatant was collected and mixed with ammonia water, then dialyzed with distilled water for 3 days, lyophilized to provide the PCCs (97 mg). Then, NAcHis was conjugated to PCCs using CDI coupling reaction. In brief, NAcHis (493 mg) was dissolved in DMSO (10 mL), then CDI (1.62 g) was added. After stirring for 4 h, the solution was evaporated to dryness, then PCCs (115 mg) dissolved in distilled water (20 mL) was added and stirred for 12 h. After dialyzed with distilled water for 3 days, the NAcHis-PCCs product (116 mg) was obtained by lyophilization. 1 H NMR and FT-IR spectra of NAcHis-PCCs were recorded on Bruker UX-500 spectrometer and Bruker Equinox-55 spectrometer, respectively. The degree of substitution (DS, defined as the number of PC per 100 sugar residues) of PC moieties was measured as described by Zeng [18], the DS (defined as the number of NAcHis per 100 sugar residues) of NAcHis moieties was determined by element analysis (PerkinElmer).
2.4. Fluorescence measurement Fluorescence spectroscopy was used to determine the critical micelle concentration (CMC) of NAcHis-PCCs using pyrene fluorescence probe method as described by Aguiar [21]. Briefly, a certain amount of pyrene was dissolved in acetone, making the final concentration 1.0 × 10−4 mg/mL, then 100 μL pyrene solution was added to each test tubes, dried under nitrogen atmosphere, and 5 mL stock solution of NAcHis-PCCs concentration ranging from 0.0001 mg/mL to 1 mg/mL was added, making the final pyrene concentration in mixed solution to be 2.0 × 10−6 mg/mL. The stock solution was equilibrated in dark under gentle shaking for 12 h at room temperature and sonicated at 100 W for 5 min before use. The fluorescence emission of pyrene was recorded at the range of 350 nm to 500 nm with the exciting wavelength of 336 nm using a fluorescence spectrophotometer (SHIMADZU RF-5301, Japan). The peak height-intensity ratios (I1/I3) of the first band (373 nm, I1) to the third band (384 nm, I3) were analyzed as a function of logarithm of NAcHis-PCCs concentration to determine the CMC value of NAcHis-PCCs.
2.5. Physicochemical characterization of nanoparticles The size and morphological characteristics of nanoparticles were investigated by atomic force microscopy (AFM). The nanoparticles were dispersed in ethanol, sonicated at 100 W for 5 min to get a better distribution, then one drop of solution was deposited on a clean silicon glass, dried under room temperature and observed using AFM (Bioscope catalyst). The mean hydrodynamic diameter, size distribution and zeta potential of nanoparticles in distilled water (final concentration: 0.1 mg/mL) were also investigated using Malvern Zetasizer Nano ZS (Malvern, UK). To investigate the effect of pH on the particle size of NAcHis-PCCs nanoparticles, NAcHis-PCCs nanoparticles were dispersed in a serial of PBS solution with pH value ranging from 7.4 to 5.0 at a concentration of 0.1 mg/mL, incubated at 37 °C for 1 h, then measured at room temperature by Malvern Zetasizer Nano ZS (Malvern, UK). All measurements were done in triplicate.
2.6. Cytotoxicity assay The cytotoxicity of NAcHis-PCCs nanoparticles was evaluated by NIH/3T3 mouse embryonic fibroblasts using a MTT cytotoxicity assay kit (Vybrant® MTT Cell proliferation Assay Kit) according to the manufacturer's manual. The measurement was done in triplicate. According to ISO 10993-5 [22], a cell viability lower than 70% in comparison to the negative control was regarded as cytotoxic.
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2.7. Hemolysis assay and RBCs morphology study Blood sampling procedures were approved by the ethics committee of Jinan University. Hemolytic activity of NAcHis-PCCs nanoparticles were rated by measuring hemolysis rate and morphology observation on rabbit red blood cells (RBCs) as described by Oda [23]. HEPES buffer was used as negative hemolysis control and 1% Triton X-100 (v/v) as positive hemolysis control. The measurement was done in triplicate. The percentage of hemolysis was calculated by the following equation: H ð%Þ ¼
A−A0 100% A100 −A0
ð1Þ
where A is the absorbance of the test sample, A0 is the absorbance of the negative control, A100 is the absorbance of the positive control. To further investigate the effect of nanoparticles on the morphology of RBCs, the RBCs were incubated with NAcHis-PCCs nanoparticles for 30 min, washed with PBS three times, and then fixed with 4% paraformaldehyde overnight. After dehydration in graded alcohols (ethanol/water (v/v) = 70%, 80%, 90%, 95%, 100%), the fixed RBCs were coated with gold and examined by scanning electron microscope (SEM, Zeiss ULTRA 55). 2.8. C3a complement assay To assess complement activation caused by NAcHis-PCCs nanoparticles, the cleavage of complement component C3 was monitored by measuring the formation of its activation product C3a peptide using a rabbit complement fragment 3a (C3a) ELISA Kit (CUSABIO Life science) according to the manufacturer's manual. Normal saline was used as negative control and L-PEI as positive control. All measurements were done in triplicate. 2.9. In vitro drug release study The in vitro release experiments were performed as described by Li [24]. Briefly, 1 mL QUE/NAcHis-PCCs nanoparticles solution (1 mg/mL) was placed in a dialysis bag (MWCO: 8000–14,000) and dialyzed against 20 mL of PBS (pH 7.4, 5.5) under gentle shaking (100 rpm) at 37 °C respectively. At predetermined intervals, 1 mL aliquot was taken out and replaced with 1 mL fresh medium, the released QUE was determined by UPLC as described above. 3. Results and discussion 3.1. Synthesis of NAcHis-PCCs Based on the combination of Atherton-Todd reaction for coupling PC and CDI coupling reaction for linking NAcHis to the amino groups of
chitosan, amphiphilic biomimetic chitosan derivative NAcHis-PCCs was successfully synthesized (Scheme 1). By changing the feeding rate of related reactant, we can adjust the DS values of PC and NAcHis moieties for optimizing the hydrophobicity/hydrophilicity balance of NAcHis-PCCs, which greatly affected their self-assembly as well as drug loading and releasing [11]. 1H NMR spectra and FT-IR spectra of chitosan, PCCs and NAcHis-PCCs were presented in Fig. 1. As shown in Fig. 1(a), the 1H NMR spectra at 293 K contained the following peaks: Chitosan (CD3COOD/D2O = 2% (v/v), δ/ppm): δ 3.05 (br, H2 of GlcN), 3.55–3.98 (m, H3, H4, H5, H6 of GlcN); PCCs (D2O, δ/ppm): δ 2.80 (br, H2 of GlcN and GlcN-PC), 3.14 (s, N+(CH3)3), 3.55–3.98 (m, N+ CH, 2H3, H4, H5, 6 H of GlcN and GlcN-PC), 4.21 (br, OPOCH),2 4.45 (br, H1 of GlcN and GlcN-PC); NAcHis-PCCs (D2O, δ/ppm): δ 2.80 (br, H2 of GlcN and GlcN-PC), δ 3.14 (s,N+(CH3)3), δ 3.55–3.98 (m, N+ CH, 2H3, H4, H5, H6 of GlcN and GlcN-PC), δ 4.21 (br, OPOCH),2 δ 4.45 (br, H1 of GlcN and GlcN-PC), δ 3.66, 4.50, 7.50 (ascribed to NAcHis moiety). The aforementioned results suggested that both PC and NAcHis groups have been successfully coupled to the chitosan backbone to form NAcHis-PCCs. The DS of PC moiety was measured by calculating the amount ratio of H of N+(CH3)3 to H2 of GlcN and GlcN-PC, while the DS of NAcHis moiety was determined by element analysis (Table S1, Support Information) since it could not be exactly calculated by comparing the ratio of NAcHis protons to glucosamine protons owing to the self-aggregation of NAcHis-PCCs conjugates in the aqueous solution. In this work, NAcHis-PCCs with DS (PC) = 42.0% and DS (NAcHis) = 12.4% was prepared for loading QUE by self-assembly mainly due to the hydrophobic interaction between NAcHis-PCCs and QUE. The chemical structure of chitosan, PCCs and NAcHis-PCCs were also analyzed using FT-IR spectroscopy, as shown in Fig. 1(b). Compared to chitosan, new peaks at 1595 cm−1, 1477 cm− 1, 1205 cm− 1 and NH, bending ofN+ (CH3) 972 cm−1 ascribed to the stretching vibration of O and the stretching ofC O P, respectively, ap3, asymmetric stretching of OP peared for PCCs, and the bending vibration ofNH2 shifted from 1637 cm−1 to 1650 cm−1, confirming the successful conjugation of PC moiety to chitosan [25]. As for NAcHis-PCCs, new peak ascribed to the stretching vibration of carbonyl group appeared at 1319 cm−1 and the bending vibration ofNH2 shifted to 1652 cm−1, providing further evidence for successful conjugation of NAcHis to chitosan backbone. 3.2. Formation and characterization of NAcHis-PCCs nanoparticles Critical micelle concentration (CMC) is an important parameter affecting the aggregation behavior of amphiphilic polymers in aqueous solution. The self-assembly behavior of NAcHis-PCCs in distilled water was investigated by fluorescence spectroscopy using pyrene as a fluorescence probe to determine its CMC value, by analyzing the peak height-intensity ratio (I1/I3) of pyrene as a function of logarithm of
Scheme 1. Synthesis route of NAcHis-PCCs.
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Fig. 1. (a) 1H NMR spectra and (b) FT-IR spectra of chitosan, PCCs and NAcHis-PCCs.
NAcHis-PCCs concentration as shown in Fig. 2(a). It was found that the CMC value of NAcHis-PCCs was 6.9 × 10−3 mg/mL, which was lower than those of low-molecular-weight surfactants, e.g., 2.3 mg/mL for sodium dodecyl sulfate (SDS) in water [21], and 0.256 mg/mL for PCCs and 1.77 × 10−2 mg/mL for DCA-PCCs with the same DS of PC moiety reported in our previous work [11,25], as well as those of some hydrophobically modified chitosan derivatives (in the magnitude of 10−2 mg/mL) [11]. It was generally accepted that the CMC value of hydrophobically modified water-soluble polymers was dependent on their hydrophilic/hydrophobic balance, and usually decreased with the increase of hydrophobicity and DS of hydrophobic pendant groups due to the enhanced driven force of hydrophobic attraction. The lower CMC value of self-assembled NAcHis-PCCs nanoparticles indicated that they might have an improved stability upon dilution in the bloodstream after intravenous administration for drug delivery application. The morphology of NAcHis-PCCs nanoparticles was observed by AFM as shown in Fig. 2(b). It could be seen that NAcHis-PCCs can selfassemble to form nanosized spherical micelles in neutral water with the particle size of about 90 nm in dry state. The mean hydrodynamic diameter of NAcHis-PCCs nanoparticles was 367 ± 2.9 nm with PDI (polydispersity indices) of 0.309 measured by dynamic light scattering (DLS) technique as shown in Fig. 2(c), indicating a relative narrow size distribution for NAcHis-PCCs nanoparticles. And the NAcHis-PCCs nanoparticles were quite stable with 24 h (Fig. S1, Support Information). Moreover, the zeta potential of NAcHis-PCCs nanoparticles was +5.35 ± 0.18 mV, which was close to the zeta potential value of selfassembled DCA-PCCs nanoparticles of 5.49 ± 0.43 mV [11], but much smaller than that of pure chitosan nanohydrogels without any chemical
cross-linker, about + 35 mV [26], due to the consumption of amino group through the conjugation of PC and NAcHis and the existence of electrically neutral zwitterionic PC groups in the nanoparticle surface. It should be noted that the formed hydrophilic PC shell might act as a biologically inert artificial cell membrane to inhibit the non-specific protein adsorption and tissue immunoreactions, and prolong the circulation time of nanoparticles in bloodstream [8]. 3.3. pH-Responsive behavior of NAcHis-PCCs nanoparticles The pH-responsive behavior of NAcHis-PCCs nanoparticles was investigated by monitoring the size change of nanoparticles in PBS solutions with different pH value with DLS measurement as shown in Fig. 3. It could be seen that the particle size was almost stable at ~ 367 nm when the pH was above 6.5, while nearly doubled to about 650 nm when the pH dropped to below 6.0. This pH-dependent behavior of particle size should be obviously attributed to the breakdown of the hydrophobic/hydrophilic balance of self-assembled nanoparticles caused by the protonation of imidazole group in NAcHis moiety at acidic pH lower than its pKa value. The similar phenomena have been also observed for the self-assembled nanoparticles based on other amphiphilic copolymers containing pH-sensitive histidine moieties. Qiu et al. reported a sudden increase in average size of self-assembled hyaluronic acid-g-poly(L-histidine) copolymer micelles from pH 6.0 to 5.0 [27], and Yang et al. reported the increase of effective diameters of selfassembled histidine-conjugated poly(amino acid) derivatives poly(2hydroxyethyl aspartamide)-g-C18-histidine nanoparticles at pH 5.0 compared to pH 7.4 [28]. The results also confirmed the successful
Fig. 2. (a) CMC value, (b) AFM image, and (c) Size distribution of NAcHis-PCCs nanoparticles.
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concentration up to 2.0 mg/mL for 24 h was about 80%. It was believed that the cationic nature of macromolecules or particles is generally associated with cytotoxic effects mainly due to their electrostatic interaction with anionic components of the cell membranes, while biomimetic PC groups can effectively shield the electrostatic interaction to improve the biocompatibility probably due to their electrically neutral zwitterionic nature and excellent hydration capability. According to ISO 10993–5, NAcHis-PCCs nanoparticles up to 2.0 mg/mL was no cytotoxic, suggesting their safety as carriers for delivery of therapeutic/diagnostic agents. 3.5. Hemolysis assay and RBC morphology
Fig. 3. Particle size of NAcHis-PCCs nanoparticles in different pH media, means ± SD (n = 3).
construction of acid-triggered nanocarriers for drug delivery. Actually, pH-responsive amphiphilic copolymer micelles could be formed by the self-assembly of amphiphilic block copolymers [24] and the hydrophobically modified water-soluble polymers (e.g. polysaccharides) [6,27,28]. The former has several advantages that their threedimensional structure, stability, and pH-responsive properties can be easily manipulated by adjusting the structure of monomers and reaction conditions, while the later can take advantage of natural polysaccharides with some unique properties such as excellent biocompatibility, non-toxicity and bioactive properties. Both of them can be used as drug delivery systems. 3.4. In vitro cytotoxicity study Low toxicity is an important requirement for materials used in pharmaceutical and biomedical applications. In this work, the in vitro cytotoxicity of NAcHis-PCCs nanoparticles to NIH/3T3 cells was evaluated using MTT assay and presented in Fig. 4(a). It could be seen that NAcHis-PCCs nanoparticles displayed a dose-dependent cytotoxicity against NIH/3T3 cells, which was in good agreement with previous reports on cytotoxicity of polycations, including trimethyl chitosan (TMC) [29], poly(ethylene imine)s (PEI) [30], DCA-PCCs [11]. And the relative viability of NIH/3T3 cells treated with NAcHis-PCCs at the
Hemolytic activity was also an important initial assessment of toxic effect of biomaterials. The percentages of hemolysis induced by NAcHis-PCCs nanoparticles as a function of their concentration were also presented in Fig. 4(a). It was found that the hemolysis rate of NAcHis-PCCs nanoparticles at the concentration range of 0.25 mg/mL to 2.0 mg/mL were all below 2%, indicating that NAcHis-PCCs nanoparticles up to 2.0 mg/mL were non-hemolytic since hemolysis rate below 2% was classified as non-hemolytic according to ASTM standard F756 [31]. Pure chitosan was known as hemolytic due to its positively charged amine group, which would interact with anionic glycoproteins presented in erythrocyte membrane and induce curvature off erythrocyte membrane, ultimately lead to membrane rupture and release hemoglobin [32]. NAcHis-PCCs nanoparticles exhibited non-hemolytic property mainly owing to the existence of biomimetic hydrophilic zwitterionic PC groups in the surface, which can effectively inhibit the damaging effects on RBCs. Additionally, the morphology of RBCs incubated with NAcHis-PCCs nanoparticles was observed by SEM as shown in Fig. 4(c). It could be seen that NAcHis-PCCs nanoparticles induced no obvious morphological change of RBCs compared to negative control group incubated with normal saline as shown in Fig. 4(b). The results confirmed that NAcHisPCCs nanoparticles were quite non-hemolytic. 3.6. C3a complement assay The complement system plays a key role for immune surveillance and homeostasis, which recognizes potential targets (microorganisms, artificial materials, apoptotic and necrotic cells, etc.), marks them for clearance and/or lysis, and initiates powerful inflammatory reactions [33,34]. It has been reported that many nanoparticles elicited moderate to severe complement activation, which greatly limited their clinic application [35]. Even PEGylated nanoparticles have been reported to
Fig. 4. (a) Cytotoxicity and hemolysis of NAcHis-PCCs nanoparticles, means ± SD (n = 3); and SEM images of RBCs (b) control and (c) incubated with NAcHis-PCCs at 1.0 mg/mL.
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Fig. 5. Formation of C3a after incubation with normal saline (NS), linear polyethyleneimine (L-PEI), as well as PCCs and NAcHis-PCCs nanoparticles at different concentration, means ± SD (n = 3).
activate complement system in a concentration and molecular weight dependent manner, though the mechanism of the complement activation induced by PEG has not been completely elucidated yet [36]. In generally, the complement system is activated via three different routes: the classical pathway (CP), the alternative pathway (AP), and the Lectin pathway (LP), and C3 would be cleaved into C3a and C3b in the activation of all three pathways under normal conditions. Thus in this work, the level of complement fragment C3a, an anaphylatoxin that initiates inflammatory pathways, was monitored to evaluate the effect of NAcHis-PCCs nanoparticles on complement activation as shown in Fig. 5. It was found that the amount of C3a generated upon both PCCs and NAcHis-PCCs nanoparticles at all the concentrations up to 2.0 mg/mL was not significantly different from that in the control normal saline. Sharma et al. have reported that PEI and chitosan carrying amino groups can adsorb C3 and in turn activate the complement system through an alternative pathway [32]. The data suggested that biomimetic PC groups can significantly suppress the extent of complement activation of chitosan, and NAcHis-PCCs nanoparticles with PC shell may prevent undesired biological response related to the complement activation, since the tightly coupled hydration PC shell of the micelles exhibited strong anti-adsorption capacity to plasma proteins including complement system proteins, and suppressed the complement activation [10].
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Fig. 7. Release profiles of QUE/NAcHis-PCCs nanoparticles at pH 5.5 and pH 7.4.
3.7. QUE loading and in vitro release study QUE was successfully loaded into the core of NAcHis-PCCs nanoparticles through hydrophobic interactions by a solvent evaporation method, and the LC and LE values of QUE were found to be 2.95% and 30.4% respectively determined by UPLC, which were slightly smaller than those of QUE-loaded DCA-PCCs nanoparticles (4.38% for LC and 46.1% for LE) prepared by the same method due to the different interaction between QUE and hydrophobic side group of amphiphilic biomimetic chitosan derivatives [11]. As shown in Fig. 6, QUE-loaded NAcHis-PCCs nanoparticles were spherical in shape with the diameter of about 170 nm in dry state larger than blank NAcHis-PCCs nanoparticles observed by AFM, while their hydrodynamic diameter determined by DLS was about 283 nm with PDI of 0.230 and zeta potential to be +5.32 ± 0.13 mV, which was smaller than blank NAcHis-PCCs nanoparticles. The results indicated that QUE-loaded NAcHis-PCCs nanoparticles almost had no change in the morphology and surface property but became more compact compared to blank nanoparticles possibly due to the hydrophobic interactions between QUE and NAcHis group. Fig. 7 showed the in vitro release profile of QUE from QUE/NAcHisPCCs nanoparticles at 37 °C under different pH condition, pH 7.4 and 5.5 corresponding to the environment of blood and mature endosomes of tumor cells respectively [37]. Both release profiles presented similar biphasic drug release kinetics with initial fast release followed by a slow release phase. About 50% of loaded QUE were released in 1 h at pH 5.5 compared with only 25% released at pH 7.4, and both release curves came to a plateau in 16 h with cumulative QUE release to be
Fig. 6. (a) AFM image, and (b) size distribution of QUE/NAcHis-PCCs nanoparticles.
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80% for pH 5.5 while only 55% for pH 7.4 respectively. The results indicated that QUE/NAcHis-PCCs nanoparticles showed an enhanced drug release behavior under acidic conditions (pH 5.5) compared to physiological conditions (pH 7.4), which consisted well with the acidtriggered behavior of NAcHis-PCCs nanoparticles measured by DLS. Actually, the mechanism of drug release from polymeric micelles was very complicated, which could be classified as diffusion-controlled, swellingcontrolled, degradation-controlled, and their combination. At pH 7.4, the release of QUE from NAcHis-PCCs micelles was slow mainly via a diffusion-controlled mechanism, while at pH 5.5, the NAcHis groups of NAcHis-PCCs were protonated, leading the swelling and demicellization of the NAcHis-PCCs micelles, which finally caused an increased release rate, followed a swelling-demicellization-releasing combination mechanism [38]. Since some pathological sites including solid tumors and inflammations usually have acidic microenvironment relative to normal physiological pH, NAcHis-PCCs nanoparticles can be used as a sitespecific drug delivery system targeted to solid tumors or inflammations for improving treatment efficacy.
[7]
[8]
[9]
[10]
[11]
[12]
4. Conclusion
[13]
Novel pH-responsive biomimetic chitosan derivative with phosphorylcholine and N-acetyl-L-histidine was synthesized via Atherton-Todd reaction and carbonyldiimidazole coupling reaction, which can self-assemble to form NAcHis-PCCs nanoparticles as a biomimetic platform for constructing intelligent drug delivery nanosystems. The in vitro biocompatibility assessment revealed that NAcHis-PCCs nanoparticles exhibited excellent biocompatibility to avoid adverse biological response, such as cytotoxicity, hemolysis and complement activation mainly due to their biomimetic PC shell, which also showed an acid-triggered behavior due to the protonation of imidazole group in NAcHis moiety at acidic pH. QUE was effectively loaded in NAcHisPCCs nanocarriers and showed an enhanced release at acidic pH 5.5 compared to neutral pH 7.4. It was supposed that pH-responsive biomimetic NAcHis-PCCs may be a promising nanocarrier for targeting drug delivery to specific sites with acidic microenvironments especially for anti-tumor or anti-inflammatory therapy.
[14]
Acknowledgement The authors would like to acknowledge financial support from the National Natural Science Foundation of China (Grant No. 31040027 and No. 31370974). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.03.099.
[15]
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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec
Novel self-assembled pH-responsive biomimetic nanocarriers for drug delivery Minming Wu, Zhaoyu Cao, Yunfei Zhao, Rong Zeng ⁎, Mei Tu, Jianhao Zhao Department of Materials Science and Engineering, College of Science and Engineering, Jinan University, Guangzhou 510632, PR China
a r t i c l e
i n f o
Article history: Received 28 July 2015 Received in revised form 8 March 2016 Accepted 29 March 2016 Available online 4 April 2016 Keywords: Biomimetic pH-responsive Chitosan Nanocarriers Controlled release
a b s t r a c t Novel pH-responsive biodegradable biomimetic nanocarriers were prepared by the self-assembly of N-acetyl-Lhistidine-phosphorylcholine-chitosan conjugate (NAcHis-PCCs), which was synthesized via Atherton-Todd reaction to couple biomembrane-like phosphorylcholine (PC) groups, and N,N′-carbonyldiimidazole (CDI) coupling reaction to link pH-responsive N-acetyl-L-histidine (NAcHis) moieties to chitosan. In vitro biological assay revealed that NAcHis-PCCs nanoparticles had excellent biocompatibility to avoid adverse biological response mainly owing to their biomimetic PC shell, and DLS results confirmed their pH-responsive behavior in acidic aqueous solution (pH ≤ 6.0). Quercetin (QUE), an anti-inflammatory, antioxidant and potential anti-tumor hydrophobic drug, was effectively loaded in NAcHis-PCCs nanocarriers and showed a pH-triggered release behavior with the enhanced QUE release at acidic pH 5.5 compared to neutral pH 7.4. The results indicated that pHresponsive biomimetic NAcHis-PCCs nanocarriers might have great potential for site-specific delivery to pathological acidic microenvironment avoiding unfavorable biological response. © 2016 Elsevier B.V. All rights reserved.
1. Introduction Stimuli-responsive polymeric nanocarriers have attracted great attention for the controlled delivery of bioactive agents like proteins, oligonucleotides, or small-molecular drugs to specific sites in vivo to enhance therapy efficacy, which can deliver and release the cargo to specific biological environment due to their abrupt physical and chemical changes triggered by specific chemical, biochemical, or physical stimuli, such as pH, enzyme, light and temperature [1,2]. In particularly, pH-responsive nanocarriers have been extensively developed for passive targeting to some pathological sites with metabolic acidic microenvironment relative to normal physiological pH (i.e., 7.4), such as solid tumors, chronic obstructive pulmonary disease (COPD), neuromuscular diseases and rheumatoid arthritis [3]; as well as improving the intracellular delivery efficacy of therapeutic/diagnostic agents due to the intracellular pH gradients (pH 5.9–6.2 in early endosomes, pH 5.0–5.5 in late endosomes and lysosomes, and neutral in cytosol) [4]. In the past decades, a number of pH-responsive polymeric nanocarriers have been successfully fabricated based on the incorporation of the moieties with pH-tunable hydrophilic/hydrophobicity including carboxyl and/or tertiary amino groups, or the pH-cleavable linkages such as hydrazone, hydrazide and acetal [2]. Lee et al. reported a pH-sensitive poly (tetrahydropyran-2-yl methacrylate) nanoparticles for local drug delivery to acidic inflammatory environments [5]. Park et al. reported pHsensitive N-acetyl histidine-conjugated glycol chitosan self-assembled ⁎ Corresponding author. E-mail address: [email protected] (R. Zeng).
http://dx.doi.org/10.1016/j.msec.2016.03.099 0928-4931/© 2016 Elsevier B.V. All rights reserved.
nanoparticles for intracytoplasmic delivery of the anticancer drug paclitaxel [6]. However, it's still a challenge to avoid unfavorable biological response in vivo, e.g. the rapid clearance by the natural defence mechanisms of the body, before they made the response triggered by acidic pH. Recently, cell membrane mimic phosphorylcholine (PC) has received significant interest to modify nanocarriers for avoiding the undesired biological response instead of the traditional poly(ethylene glycol) functionalization, since recent papers reported some unfavorable biological responses related to PEGylated nanocarriers, including accelerated blood clearance (ABC) phenomenon, hypersensitivity and inhibiting cellular uptake [7], while PC has excellent hemocompatibility and protein-resistant property mainly due to its strong hydration capacity via electrostatic interactions [8], and PC-based nanosystems could enter living cells through fusogenic interaction with the plasma membrane [9]. Zhao et al. reported that polymer micelles bearing cell outer membrane phosphorylcholine can minimize reticuloendothelial system clearance and circulate for long time to reach the target [10]. In our previous work, we have successfully constructed bio-inspired nanocarriers with excellent biocompatibility based on the self-assembly of biodegradable amphiphilic biomimetic deoxycholic acid (DCA)-PC-chitosan derivative for hydrophobic drug or protein delivery [11,12]. Quercetin (QUE), a well-known anti-inflammatory, antioxidant and potential anti-tumor flavonoid drug, has broad employments in biological, pharmacological and medical benefits [13,14]. It has been found that QUE played important roles in several anti-cancer therapies, such as treatment of human breast cancer, lung cancer, and cervical cancer in ways of antioxidative activities, inhibition of cell circle proliferation
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and enzymes activities [15–17]. In spite of the numerous advantages QUE bears, the clinical application of QUE was restrained by its poor water-solubility and bioavailability. In this work, we reported a novel approach to construct pH-responsive biomimetic nanocarriers for sitespecific delivery of QUE to tumor or inflammatory acidic microenvironment based on the self-assembly of biodegradable biomimetic chitosan derivative, N-acetyl-L-histidine-phosphorylcholine-chitosan (NAcHisPCCs), which possessed hydrophilic PC groups to avoid the undesired biological response caused by non-specific protein adsorption, and Nacetyl-L-histidine (NAcHis) groups as pH-responsive moieties, since the pKa of the imidazole group in histidine is around 6.5, which would be protonated to be hydrophilic to destabilize nanocarriers and eventually release cargo when milieu pH was below 6.5 [6]. The physicochemical properties, in vitro biocompatibility and pH-dependent drug release behavior of nanocarriers were investigated.
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2.3. Preparation of NAcHis-PCCs and QUE/NAcHis-PCCs nanoparticles Blank NAcHis-PCCs and QUE/NAcHis-PCCs nanoparticles were prepared by a solvent evaporation method [20]. Briefly, NAcHis-PCCs (10 mg) was dissolved in H2O (1 mL), diluted with ethanol (2 mL), and mixed with 1 mg QUE or not, and then rotate evaporated at 40 °C to obtain a thin film. After addition of 10 mL H2O, the solution was gently stirred for 5 min, filtered through a 0.45 μm Millipore filter and lyophilized to obtain the nanoparticles. The QUE loading efficiency (LE, amount of QUE loaded in nanoparticles/total amount of QUE in added solution) and loading capacity (LC, amount of QUE loaded in nanoparticles/mass of QUE-loaded nanoparticles) of NAcHis-PCCs nanoparticles were evaluated using UPLC (Acquity UPLC Waters, US, detection wavelength: 374 nm, BEH-Phenyl column, Eluent: 0.1% formic acid in water: methanol (40:60 v/v), Flow rate: 0.4 mL/min) as described in our previous work [11].
2. Experimental 2.1. Materials Chitosan (Mw = 29 k, Deacetylation degree: 100%) was obtained by repeating N-deacetylation of chitosan (Brookfield viscosity: 20 cps, Sigma-Aldrich). Diphenyl phosphite, pyrene and choline chloride was obtained from Acros Organics. L-PEI (Mw = 25 k) was purchased from Alfa-Aesar. N-acetyl-L-histidine monohydrate (NAcHis), N,N ′-carbonyldiimidazole (CDI) and quercetin (QUE) were purchased from Aladdin Reagent Co., Ltd. Other chemicals and solvents were of analytical grade. 2.2. Synthesis of NAcHis-PCCs All experiments involving water-sensitive compounds were conducted under dry conditions. Firstly, PCCs was synthesized according to our previous work [18]. Chitosan with deacetylation degree of 100% was obtained by treatment with 40% (w/v) NaOH aqueous solution at 110 °C for 1.5 h three times, and confirmed by 1H NMR, then modified to 6-O-trityl chitosan according to Nishimura's method [19]. 200 mg (0.5 mmol of free NH2) 6-O-trityl chitosan was dissolved in a mixed solution of dimethylacetamide (DMA, 10.0 mL), triethylamine (1.05 mL, 7.5 mmol) and tetrachloromethane (0.475 mL, 5.0 mmol) (Solution A). 740 mg (5.3 mmol) of choline chloride was dissolved in 20.0 mL of freshly distilled pyridine/DMSO (1:10) at room temperature, then 0.475 mL (2.5 mmol) of diphenyl phosphite was added and stirred for 2 h. After evaporation, the crude product of dicholinyl H-phosphonate dichloride was dissolved in 15 mL of isopropanol (Solution B). Solution B was added dropwise to solution A in ice-water bath, stirred for 12 h, then the resulting solution was evaporated to dryness. 15 mL of formic acid was added and stirred for 1 h under room temperature, then rotary evaporation was used to remove the remaining formic acid. The residue was dissolved in distilled water (20 mL) and filtered, supernatant was collected and mixed with ammonia water, then dialyzed with distilled water for 3 days, lyophilized to provide the PCCs (97 mg). Then, NAcHis was conjugated to PCCs using CDI coupling reaction. In brief, NAcHis (493 mg) was dissolved in DMSO (10 mL), then CDI (1.62 g) was added. After stirring for 4 h, the solution was evaporated to dryness, then PCCs (115 mg) dissolved in distilled water (20 mL) was added and stirred for 12 h. After dialyzed with distilled water for 3 days, the NAcHis-PCCs product (116 mg) was obtained by lyophilization. 1 H NMR and FT-IR spectra of NAcHis-PCCs were recorded on Bruker UX-500 spectrometer and Bruker Equinox-55 spectrometer, respectively. The degree of substitution (DS, defined as the number of PC per 100 sugar residues) of PC moieties was measured as described by Zeng [18], the DS (defined as the number of NAcHis per 100 sugar residues) of NAcHis moieties was determined by element analysis (PerkinElmer).
2.4. Fluorescence measurement Fluorescence spectroscopy was used to determine the critical micelle concentration (CMC) of NAcHis-PCCs using pyrene fluorescence probe method as described by Aguiar [21]. Briefly, a certain amount of pyrene was dissolved in acetone, making the final concentration 1.0 × 10−4 mg/mL, then 100 μL pyrene solution was added to each test tubes, dried under nitrogen atmosphere, and 5 mL stock solution of NAcHis-PCCs concentration ranging from 0.0001 mg/mL to 1 mg/mL was added, making the final pyrene concentration in mixed solution to be 2.0 × 10−6 mg/mL. The stock solution was equilibrated in dark under gentle shaking for 12 h at room temperature and sonicated at 100 W for 5 min before use. The fluorescence emission of pyrene was recorded at the range of 350 nm to 500 nm with the exciting wavelength of 336 nm using a fluorescence spectrophotometer (SHIMADZU RF-5301, Japan). The peak height-intensity ratios (I1/I3) of the first band (373 nm, I1) to the third band (384 nm, I3) were analyzed as a function of logarithm of NAcHis-PCCs concentration to determine the CMC value of NAcHis-PCCs.
2.5. Physicochemical characterization of nanoparticles The size and morphological characteristics of nanoparticles were investigated by atomic force microscopy (AFM). The nanoparticles were dispersed in ethanol, sonicated at 100 W for 5 min to get a better distribution, then one drop of solution was deposited on a clean silicon glass, dried under room temperature and observed using AFM (Bioscope catalyst). The mean hydrodynamic diameter, size distribution and zeta potential of nanoparticles in distilled water (final concentration: 0.1 mg/mL) were also investigated using Malvern Zetasizer Nano ZS (Malvern, UK). To investigate the effect of pH on the particle size of NAcHis-PCCs nanoparticles, NAcHis-PCCs nanoparticles were dispersed in a serial of PBS solution with pH value ranging from 7.4 to 5.0 at a concentration of 0.1 mg/mL, incubated at 37 °C for 1 h, then measured at room temperature by Malvern Zetasizer Nano ZS (Malvern, UK). All measurements were done in triplicate.
2.6. Cytotoxicity assay The cytotoxicity of NAcHis-PCCs nanoparticles was evaluated by NIH/3T3 mouse embryonic fibroblasts using a MTT cytotoxicity assay kit (Vybrant® MTT Cell proliferation Assay Kit) according to the manufacturer's manual. The measurement was done in triplicate. According to ISO 10993-5 [22], a cell viability lower than 70% in comparison to the negative control was regarded as cytotoxic.
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2.7. Hemolysis assay and RBCs morphology study Blood sampling procedures were approved by the ethics committee of Jinan University. Hemolytic activity of NAcHis-PCCs nanoparticles were rated by measuring hemolysis rate and morphology observation on rabbit red blood cells (RBCs) as described by Oda [23]. HEPES buffer was used as negative hemolysis control and 1% Triton X-100 (v/v) as positive hemolysis control. The measurement was done in triplicate. The percentage of hemolysis was calculated by the following equation: H ð%Þ ¼
A−A0 100% A100 −A0
ð1Þ
where A is the absorbance of the test sample, A0 is the absorbance of the negative control, A100 is the absorbance of the positive control. To further investigate the effect of nanoparticles on the morphology of RBCs, the RBCs were incubated with NAcHis-PCCs nanoparticles for 30 min, washed with PBS three times, and then fixed with 4% paraformaldehyde overnight. After dehydration in graded alcohols (ethanol/water (v/v) = 70%, 80%, 90%, 95%, 100%), the fixed RBCs were coated with gold and examined by scanning electron microscope (SEM, Zeiss ULTRA 55). 2.8. C3a complement assay To assess complement activation caused by NAcHis-PCCs nanoparticles, the cleavage of complement component C3 was monitored by measuring the formation of its activation product C3a peptide using a rabbit complement fragment 3a (C3a) ELISA Kit (CUSABIO Life science) according to the manufacturer's manual. Normal saline was used as negative control and L-PEI as positive control. All measurements were done in triplicate. 2.9. In vitro drug release study The in vitro release experiments were performed as described by Li [24]. Briefly, 1 mL QUE/NAcHis-PCCs nanoparticles solution (1 mg/mL) was placed in a dialysis bag (MWCO: 8000–14,000) and dialyzed against 20 mL of PBS (pH 7.4, 5.5) under gentle shaking (100 rpm) at 37 °C respectively. At predetermined intervals, 1 mL aliquot was taken out and replaced with 1 mL fresh medium, the released QUE was determined by UPLC as described above. 3. Results and discussion 3.1. Synthesis of NAcHis-PCCs Based on the combination of Atherton-Todd reaction for coupling PC and CDI coupling reaction for linking NAcHis to the amino groups of
chitosan, amphiphilic biomimetic chitosan derivative NAcHis-PCCs was successfully synthesized (Scheme 1). By changing the feeding rate of related reactant, we can adjust the DS values of PC and NAcHis moieties for optimizing the hydrophobicity/hydrophilicity balance of NAcHis-PCCs, which greatly affected their self-assembly as well as drug loading and releasing [11]. 1H NMR spectra and FT-IR spectra of chitosan, PCCs and NAcHis-PCCs were presented in Fig. 1. As shown in Fig. 1(a), the 1H NMR spectra at 293 K contained the following peaks: Chitosan (CD3COOD/D2O = 2% (v/v), δ/ppm): δ 3.05 (br, H2 of GlcN), 3.55–3.98 (m, H3, H4, H5, H6 of GlcN); PCCs (D2O, δ/ppm): δ 2.80 (br, H2 of GlcN and GlcN-PC), 3.14 (s, N+(CH3)3), 3.55–3.98 (m, N+ CH, 2H3, H4, H5, 6 H of GlcN and GlcN-PC), 4.21 (br, OPOCH),2 4.45 (br, H1 of GlcN and GlcN-PC); NAcHis-PCCs (D2O, δ/ppm): δ 2.80 (br, H2 of GlcN and GlcN-PC), δ 3.14 (s,N+(CH3)3), δ 3.55–3.98 (m, N+ CH, 2H3, H4, H5, H6 of GlcN and GlcN-PC), δ 4.21 (br, OPOCH),2 δ 4.45 (br, H1 of GlcN and GlcN-PC), δ 3.66, 4.50, 7.50 (ascribed to NAcHis moiety). The aforementioned results suggested that both PC and NAcHis groups have been successfully coupled to the chitosan backbone to form NAcHis-PCCs. The DS of PC moiety was measured by calculating the amount ratio of H of N+(CH3)3 to H2 of GlcN and GlcN-PC, while the DS of NAcHis moiety was determined by element analysis (Table S1, Support Information) since it could not be exactly calculated by comparing the ratio of NAcHis protons to glucosamine protons owing to the self-aggregation of NAcHis-PCCs conjugates in the aqueous solution. In this work, NAcHis-PCCs with DS (PC) = 42.0% and DS (NAcHis) = 12.4% was prepared for loading QUE by self-assembly mainly due to the hydrophobic interaction between NAcHis-PCCs and QUE. The chemical structure of chitosan, PCCs and NAcHis-PCCs were also analyzed using FT-IR spectroscopy, as shown in Fig. 1(b). Compared to chitosan, new peaks at 1595 cm−1, 1477 cm− 1, 1205 cm− 1 and NH, bending ofN+ (CH3) 972 cm−1 ascribed to the stretching vibration of O and the stretching ofC O P, respectively, ap3, asymmetric stretching of OP peared for PCCs, and the bending vibration ofNH2 shifted from 1637 cm−1 to 1650 cm−1, confirming the successful conjugation of PC moiety to chitosan [25]. As for NAcHis-PCCs, new peak ascribed to the stretching vibration of carbonyl group appeared at 1319 cm−1 and the bending vibration ofNH2 shifted to 1652 cm−1, providing further evidence for successful conjugation of NAcHis to chitosan backbone. 3.2. Formation and characterization of NAcHis-PCCs nanoparticles Critical micelle concentration (CMC) is an important parameter affecting the aggregation behavior of amphiphilic polymers in aqueous solution. The self-assembly behavior of NAcHis-PCCs in distilled water was investigated by fluorescence spectroscopy using pyrene as a fluorescence probe to determine its CMC value, by analyzing the peak height-intensity ratio (I1/I3) of pyrene as a function of logarithm of
Scheme 1. Synthesis route of NAcHis-PCCs.
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Fig. 1. (a) 1H NMR spectra and (b) FT-IR spectra of chitosan, PCCs and NAcHis-PCCs.
NAcHis-PCCs concentration as shown in Fig. 2(a). It was found that the CMC value of NAcHis-PCCs was 6.9 × 10−3 mg/mL, which was lower than those of low-molecular-weight surfactants, e.g., 2.3 mg/mL for sodium dodecyl sulfate (SDS) in water [21], and 0.256 mg/mL for PCCs and 1.77 × 10−2 mg/mL for DCA-PCCs with the same DS of PC moiety reported in our previous work [11,25], as well as those of some hydrophobically modified chitosan derivatives (in the magnitude of 10−2 mg/mL) [11]. It was generally accepted that the CMC value of hydrophobically modified water-soluble polymers was dependent on their hydrophilic/hydrophobic balance, and usually decreased with the increase of hydrophobicity and DS of hydrophobic pendant groups due to the enhanced driven force of hydrophobic attraction. The lower CMC value of self-assembled NAcHis-PCCs nanoparticles indicated that they might have an improved stability upon dilution in the bloodstream after intravenous administration for drug delivery application. The morphology of NAcHis-PCCs nanoparticles was observed by AFM as shown in Fig. 2(b). It could be seen that NAcHis-PCCs can selfassemble to form nanosized spherical micelles in neutral water with the particle size of about 90 nm in dry state. The mean hydrodynamic diameter of NAcHis-PCCs nanoparticles was 367 ± 2.9 nm with PDI (polydispersity indices) of 0.309 measured by dynamic light scattering (DLS) technique as shown in Fig. 2(c), indicating a relative narrow size distribution for NAcHis-PCCs nanoparticles. And the NAcHis-PCCs nanoparticles were quite stable with 24 h (Fig. S1, Support Information). Moreover, the zeta potential of NAcHis-PCCs nanoparticles was +5.35 ± 0.18 mV, which was close to the zeta potential value of selfassembled DCA-PCCs nanoparticles of 5.49 ± 0.43 mV [11], but much smaller than that of pure chitosan nanohydrogels without any chemical
cross-linker, about + 35 mV [26], due to the consumption of amino group through the conjugation of PC and NAcHis and the existence of electrically neutral zwitterionic PC groups in the nanoparticle surface. It should be noted that the formed hydrophilic PC shell might act as a biologically inert artificial cell membrane to inhibit the non-specific protein adsorption and tissue immunoreactions, and prolong the circulation time of nanoparticles in bloodstream [8]. 3.3. pH-Responsive behavior of NAcHis-PCCs nanoparticles The pH-responsive behavior of NAcHis-PCCs nanoparticles was investigated by monitoring the size change of nanoparticles in PBS solutions with different pH value with DLS measurement as shown in Fig. 3. It could be seen that the particle size was almost stable at ~ 367 nm when the pH was above 6.5, while nearly doubled to about 650 nm when the pH dropped to below 6.0. This pH-dependent behavior of particle size should be obviously attributed to the breakdown of the hydrophobic/hydrophilic balance of self-assembled nanoparticles caused by the protonation of imidazole group in NAcHis moiety at acidic pH lower than its pKa value. The similar phenomena have been also observed for the self-assembled nanoparticles based on other amphiphilic copolymers containing pH-sensitive histidine moieties. Qiu et al. reported a sudden increase in average size of self-assembled hyaluronic acid-g-poly(L-histidine) copolymer micelles from pH 6.0 to 5.0 [27], and Yang et al. reported the increase of effective diameters of selfassembled histidine-conjugated poly(amino acid) derivatives poly(2hydroxyethyl aspartamide)-g-C18-histidine nanoparticles at pH 5.0 compared to pH 7.4 [28]. The results also confirmed the successful
Fig. 2. (a) CMC value, (b) AFM image, and (c) Size distribution of NAcHis-PCCs nanoparticles.
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concentration up to 2.0 mg/mL for 24 h was about 80%. It was believed that the cationic nature of macromolecules or particles is generally associated with cytotoxic effects mainly due to their electrostatic interaction with anionic components of the cell membranes, while biomimetic PC groups can effectively shield the electrostatic interaction to improve the biocompatibility probably due to their electrically neutral zwitterionic nature and excellent hydration capability. According to ISO 10993–5, NAcHis-PCCs nanoparticles up to 2.0 mg/mL was no cytotoxic, suggesting their safety as carriers for delivery of therapeutic/diagnostic agents. 3.5. Hemolysis assay and RBC morphology
Fig. 3. Particle size of NAcHis-PCCs nanoparticles in different pH media, means ± SD (n = 3).
construction of acid-triggered nanocarriers for drug delivery. Actually, pH-responsive amphiphilic copolymer micelles could be formed by the self-assembly of amphiphilic block copolymers [24] and the hydrophobically modified water-soluble polymers (e.g. polysaccharides) [6,27,28]. The former has several advantages that their threedimensional structure, stability, and pH-responsive properties can be easily manipulated by adjusting the structure of monomers and reaction conditions, while the later can take advantage of natural polysaccharides with some unique properties such as excellent biocompatibility, non-toxicity and bioactive properties. Both of them can be used as drug delivery systems. 3.4. In vitro cytotoxicity study Low toxicity is an important requirement for materials used in pharmaceutical and biomedical applications. In this work, the in vitro cytotoxicity of NAcHis-PCCs nanoparticles to NIH/3T3 cells was evaluated using MTT assay and presented in Fig. 4(a). It could be seen that NAcHis-PCCs nanoparticles displayed a dose-dependent cytotoxicity against NIH/3T3 cells, which was in good agreement with previous reports on cytotoxicity of polycations, including trimethyl chitosan (TMC) [29], poly(ethylene imine)s (PEI) [30], DCA-PCCs [11]. And the relative viability of NIH/3T3 cells treated with NAcHis-PCCs at the
Hemolytic activity was also an important initial assessment of toxic effect of biomaterials. The percentages of hemolysis induced by NAcHis-PCCs nanoparticles as a function of their concentration were also presented in Fig. 4(a). It was found that the hemolysis rate of NAcHis-PCCs nanoparticles at the concentration range of 0.25 mg/mL to 2.0 mg/mL were all below 2%, indicating that NAcHis-PCCs nanoparticles up to 2.0 mg/mL were non-hemolytic since hemolysis rate below 2% was classified as non-hemolytic according to ASTM standard F756 [31]. Pure chitosan was known as hemolytic due to its positively charged amine group, which would interact with anionic glycoproteins presented in erythrocyte membrane and induce curvature off erythrocyte membrane, ultimately lead to membrane rupture and release hemoglobin [32]. NAcHis-PCCs nanoparticles exhibited non-hemolytic property mainly owing to the existence of biomimetic hydrophilic zwitterionic PC groups in the surface, which can effectively inhibit the damaging effects on RBCs. Additionally, the morphology of RBCs incubated with NAcHis-PCCs nanoparticles was observed by SEM as shown in Fig. 4(c). It could be seen that NAcHis-PCCs nanoparticles induced no obvious morphological change of RBCs compared to negative control group incubated with normal saline as shown in Fig. 4(b). The results confirmed that NAcHisPCCs nanoparticles were quite non-hemolytic. 3.6. C3a complement assay The complement system plays a key role for immune surveillance and homeostasis, which recognizes potential targets (microorganisms, artificial materials, apoptotic and necrotic cells, etc.), marks them for clearance and/or lysis, and initiates powerful inflammatory reactions [33,34]. It has been reported that many nanoparticles elicited moderate to severe complement activation, which greatly limited their clinic application [35]. Even PEGylated nanoparticles have been reported to
Fig. 4. (a) Cytotoxicity and hemolysis of NAcHis-PCCs nanoparticles, means ± SD (n = 3); and SEM images of RBCs (b) control and (c) incubated with NAcHis-PCCs at 1.0 mg/mL.
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Fig. 5. Formation of C3a after incubation with normal saline (NS), linear polyethyleneimine (L-PEI), as well as PCCs and NAcHis-PCCs nanoparticles at different concentration, means ± SD (n = 3).
activate complement system in a concentration and molecular weight dependent manner, though the mechanism of the complement activation induced by PEG has not been completely elucidated yet [36]. In generally, the complement system is activated via three different routes: the classical pathway (CP), the alternative pathway (AP), and the Lectin pathway (LP), and C3 would be cleaved into C3a and C3b in the activation of all three pathways under normal conditions. Thus in this work, the level of complement fragment C3a, an anaphylatoxin that initiates inflammatory pathways, was monitored to evaluate the effect of NAcHis-PCCs nanoparticles on complement activation as shown in Fig. 5. It was found that the amount of C3a generated upon both PCCs and NAcHis-PCCs nanoparticles at all the concentrations up to 2.0 mg/mL was not significantly different from that in the control normal saline. Sharma et al. have reported that PEI and chitosan carrying amino groups can adsorb C3 and in turn activate the complement system through an alternative pathway [32]. The data suggested that biomimetic PC groups can significantly suppress the extent of complement activation of chitosan, and NAcHis-PCCs nanoparticles with PC shell may prevent undesired biological response related to the complement activation, since the tightly coupled hydration PC shell of the micelles exhibited strong anti-adsorption capacity to plasma proteins including complement system proteins, and suppressed the complement activation [10].
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Fig. 7. Release profiles of QUE/NAcHis-PCCs nanoparticles at pH 5.5 and pH 7.4.
3.7. QUE loading and in vitro release study QUE was successfully loaded into the core of NAcHis-PCCs nanoparticles through hydrophobic interactions by a solvent evaporation method, and the LC and LE values of QUE were found to be 2.95% and 30.4% respectively determined by UPLC, which were slightly smaller than those of QUE-loaded DCA-PCCs nanoparticles (4.38% for LC and 46.1% for LE) prepared by the same method due to the different interaction between QUE and hydrophobic side group of amphiphilic biomimetic chitosan derivatives [11]. As shown in Fig. 6, QUE-loaded NAcHis-PCCs nanoparticles were spherical in shape with the diameter of about 170 nm in dry state larger than blank NAcHis-PCCs nanoparticles observed by AFM, while their hydrodynamic diameter determined by DLS was about 283 nm with PDI of 0.230 and zeta potential to be +5.32 ± 0.13 mV, which was smaller than blank NAcHis-PCCs nanoparticles. The results indicated that QUE-loaded NAcHis-PCCs nanoparticles almost had no change in the morphology and surface property but became more compact compared to blank nanoparticles possibly due to the hydrophobic interactions between QUE and NAcHis group. Fig. 7 showed the in vitro release profile of QUE from QUE/NAcHisPCCs nanoparticles at 37 °C under different pH condition, pH 7.4 and 5.5 corresponding to the environment of blood and mature endosomes of tumor cells respectively [37]. Both release profiles presented similar biphasic drug release kinetics with initial fast release followed by a slow release phase. About 50% of loaded QUE were released in 1 h at pH 5.5 compared with only 25% released at pH 7.4, and both release curves came to a plateau in 16 h with cumulative QUE release to be
Fig. 6. (a) AFM image, and (b) size distribution of QUE/NAcHis-PCCs nanoparticles.
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80% for pH 5.5 while only 55% for pH 7.4 respectively. The results indicated that QUE/NAcHis-PCCs nanoparticles showed an enhanced drug release behavior under acidic conditions (pH 5.5) compared to physiological conditions (pH 7.4), which consisted well with the acidtriggered behavior of NAcHis-PCCs nanoparticles measured by DLS. Actually, the mechanism of drug release from polymeric micelles was very complicated, which could be classified as diffusion-controlled, swellingcontrolled, degradation-controlled, and their combination. At pH 7.4, the release of QUE from NAcHis-PCCs micelles was slow mainly via a diffusion-controlled mechanism, while at pH 5.5, the NAcHis groups of NAcHis-PCCs were protonated, leading the swelling and demicellization of the NAcHis-PCCs micelles, which finally caused an increased release rate, followed a swelling-demicellization-releasing combination mechanism [38]. Since some pathological sites including solid tumors and inflammations usually have acidic microenvironment relative to normal physiological pH, NAcHis-PCCs nanoparticles can be used as a sitespecific drug delivery system targeted to solid tumors or inflammations for improving treatment efficacy.
[7]
[8]
[9]
[10]
[11]
[12]
4. Conclusion
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Novel pH-responsive biomimetic chitosan derivative with phosphorylcholine and N-acetyl-L-histidine was synthesized via Atherton-Todd reaction and carbonyldiimidazole coupling reaction, which can self-assemble to form NAcHis-PCCs nanoparticles as a biomimetic platform for constructing intelligent drug delivery nanosystems. The in vitro biocompatibility assessment revealed that NAcHis-PCCs nanoparticles exhibited excellent biocompatibility to avoid adverse biological response, such as cytotoxicity, hemolysis and complement activation mainly due to their biomimetic PC shell, which also showed an acid-triggered behavior due to the protonation of imidazole group in NAcHis moiety at acidic pH. QUE was effectively loaded in NAcHisPCCs nanocarriers and showed an enhanced release at acidic pH 5.5 compared to neutral pH 7.4. It was supposed that pH-responsive biomimetic NAcHis-PCCs may be a promising nanocarrier for targeting drug delivery to specific sites with acidic microenvironments especially for anti-tumor or anti-inflammatory therapy.
[14]
Acknowledgement The authors would like to acknowledge financial support from the National Natural Science Foundation of China (Grant No. 31040027 and No. 31370974). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.msec.2016.03.099.
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