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Sensitive complex micelles based on host-guest recognition from chitosan-graft-β-cyclodextrin for drug release
Accepted Manuscript Title: Sensitive complex micelles based on host-guest recognition from chitosan-graft--cyclodextrin for drug release Authors: Yurong Gao, Guiying Li, Zaishuai Zhou, Lingling Gao, Qian Tao PII: DOI: Reference:
S0141-8130(17)32368-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.120 BIOMAC 7800
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-2-2017 18-5-2017 29-6-2017
Please cite this article as: Yurong Gao, Guiying Li, Zaishuai Zhou, Lingling Gao, Qian Tao, Sensitive complex micelles based on host-guest recognition from chitosan-graft--cyclodextrin for drug release, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sensitive complex micelles based on host-guest recognition from chitosan-graft-β-cyclodextrin for drug release Yurong Gao, Guiying Li, Zaishuai Zhou, Lingling Gao, Qian Tao School of Chemistry and Materials Science, Ludong University, Yantai 264025, China
Corresponding author.
E-mail address: [email protected] (G. Li).
Abstract: In this paper, pH-sensitive complex micelles were developed based on the host-guest
recognition
from
chitosan-graft-β-cyclodextrin
(CS-g-CD)
and
benzimidazole-terminated poly(ε-caprolactone) (BM-PCL) for controlled drug release. The formation and characterization of complex micelles were confirmed by fourier-transform
infrared
spectroscopy
(FTIR),
X-ray
diffraction
(XRD),
transmission electron microscopy (TEM) and laser particle analyzer. The size of complex micelles was about 200 nm with the core formed by BM-PCL/β-CD and the shell composed of chitosan. Doxorubicin (DOX), a model anticancer drug, was effectively loaded into the complex micelles via hydrophobic interactions. The encapsulation efficiency of DOX was up to 75 %. The release of DOX from complex micelles was suppressed at neutral pH solutions due to the stability of micelles but accelerated at acidic solutions and high temperatures. These sensitive complex micelles might possess potential applications as intelligent nanocarriers for anticancer drug delivery. Key words: Benzimidazole, Chitosan-graft-β-cyclodextrin, Controlled release,
1
Host-guest recognition, Sensitive polymer micelles 1. Introduction In recent years, nanoparticles have been received extensive investigations due to their potential applications in diverse areas, such as gene and drug delivery, biological sensors, hydrogen storage materials, and so on [1-3]. Among these nanoparticles, polymer micelles aggregated from amphiphilic copolymers are characterized by core-shell structure with the core formed by hydrophobic blocks and the shell formed by hydrophilic blocks. The micelles have been emerged as potential nanocarriers which the inner core can be used to encapsulate hydrophobic drugs and the outer shell offer attractive characteristics such as suitable size, low toxicity for human body, etc [4-7]. Compared with conventional polymer micelles, environmentally sensitive polymer micelles, which show intelligent response to environmental changes, such as pH, temperature, ionic strength, and so on, are most intensively studied in drug delivery systems. Sensitive polymer micelles have distinct properties including high encapsulation efficiency and drug loading, stimuli-responsive property and controlled release character, etc [8-10]. Owing to the fact that different part in human body has different pH environments, pH-sensitive polymer micelles are the best candidate for preparation of drug carriers. The pH of human body is maintained at about 7.4 in normal extracellular matrices and blood, while the solid tumors always present as acidic condition (pH ~6.0) due to the inefficient consumption of glucose into energy in cancer cells producing a large amount of lactic acid [11,12]. Therefore, pH-sensitive polymer nanocarriers have been developed for tumor targeting and 2
intracellular drug release [13]. Cyclodextrins (CDs), a series of natural macrocyclic oligosaccharides with a hydrophilic exterior and a hydrophobic interior cavity, have been extensively used in pharmaceutical field [14-17]. The hydrophobic internal cavity of CDs gives them inclusion capacity with large quantities of guest molecules, such as various poorly water-soluble drugs. Therefore, CDs have been utilized to encapsulate drugs through host-guest interactions in drug delivery system [18,19]. Such encapsulation may immensely increase the solubility of lipophilic drugs, improve the stability of labile drugs, alleviate the local and systemic toxicity, and control the drug release profiles [20-22]. For example, Tu et al. synthesized supramolecular star-shaped ABC terpolymer via the molecular recognition between β-CD based diblock copolymer poly(dimethylaminoethyl modified
methyl
pH-responsive
methacrylate)-poly(ethylene
methacrylate supramolecular
benzimidazole-terminated
(AD-PMMA)
[23].
amphiphilic
poly(ethylene
glycol)
and
Zhang et micelles
glycol)
adamantane al.
reported
based
(BM-PEG)
on and
β-cyclodextrin-modified poly(L-lactide) (CD-PLLA) by using host-guest interactions between benzimidazole and β-cyclodextrin [24]. It is well known that the host-guest interactions between β-cyclodextrin (β-CD) and benzimidazole (BM) exhibit pH-sensitivity [25]. Under the neutral conditions, the BM molecule is hydrophobic, which can be bound to β-CD cavity by host-guest interactions. The nitrogen atoms of BM are protonated under acidic conditions, which result in the dissociation of complexation between β-CD and BM [13,26]. By 3
adjusting the pH of solutions, the assembly-disassembly between BM and β-CD can be controlled. Chitosan (CS), which shows several biological properties, such as extremely nontoxicity, biodegradability, and excellent biocompatibility, is also well known for pH sensitive polymers due to the existence of -NH2 groups [10]. Combining the advantages of cyclodextrin with chitosan is conducive to improve the drug loading and pH-sensitivity of nanocarriers. Based on this conception, we designed sensitive polymer complex micelles due to the host-guest recognitions between chitosan graft β-cyclodextrin (CS-g-CD) and benzimidazole-terminated poly(ε-caprolactone) (BM-PCL). The micelles had a core-shell structure with the core formed from BM-PCL and β-CD via host-guest interactions and the shell composed of chitosan chains as shown in Scheme 1. The structure, morphology and particle size of micelles were analyzed using fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and laser particle analyzer. Doxorubicin (DOX), an anthracycline anticancer drug, was chosen as a model drug to access the applications of CS-g-CD/BM-PCL complex micelles in drug delivery. 2. Experimental 2.1. Materials Chitosan (CS) (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) with a deacetylation degree of 95 % and a viscosity-average molecular weight of 2.0 × 105 g/mol was purified by solubilization in aqueous acetic acid and reprecipitation by aqueous NaOH. β-Cyclodextrin (Shanghai Chemical Reagent Company, China) was 4
recrystallized twice from water and dried in vacuum at 80 ℃. p-Toluenesulfonyl chloride was provided from Damao Chemical Reagent Co. Ltd. (Tianjin, China). 2-Bromorthanol, ε-caprolactone (ε-CL), benzimidazole (BM), stannous octoate (Sn(Oct)2) and N,N-diisopropylethylamine (DIEA) were purchased from Shanghai Macklin
Biochemical
Co.
Ltd.
(China).
Doxorubicin
(DOX)
and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) were purchased from J&K Chemical Ltd. (Beijing, China). All other reagents used were commercially available and were of analytical grade. Double-distilled water was used for the preparation of all solutions in this study. 2.2. Preparation of copolymers 2.2.1. Preparation of chitosan-graft-β-cyclodextrin (CS-g-CD) CS-g-CD
was
synthesized
from
CS
and
mono-6-deoxy-6-(p-toluenesulfonyl)-β-cyclodextrin (6-OTs-β-CD) as described in previous reports [16,27]. Briefly, β-CD (15.0 g, 13.2 mmol) was suspended in 125.0 mL of distilled water, and then NaOH solution (5 mL, 41.1 mmol) was slowly dripped into the solution. p-Toluenesulfonyl chloride (2.5 g, 13.2 mmol) in 7.5 mL of acetonitrile was cautiously dripped into the above solution, causing immediate formation of a white sediment. After 3 h of stirring at 5 ℃, the filtrate was collected by filtration. The pH of solution was adjusted to 6.0 by hydrochloric acid, and then the solution was placed in the refrigerator overnight at 4 ℃. The precipitation of 6-OTs-β-CD was recrystallized twice from water and dried in vacuum at 50 ℃. Then, CS (0.5 g) was dissolved in 1.0 % acetic acid aqueous solution (pH 3.0, 40 mL). The 5
6-OTs-β-CD (0.5 g) was dissolved in 20 mL N,N-dimethylformamide (DMF) before it was added into CS solution. The reaction mixture was refluxed at 100 ℃ for 14 h after purging with nitrogen. The raw products CS-g-CD were purified by soxhlets using acetone for 48 h and then dried in vacuum. The yield of CS-g-CD was calculated to be 65.58 % by the gravimetric analysis. 2.2.2. Preparation of BM-PCL PCL-Br was synthesized by ring-opening polymerization of ε-caprolactone monomer using 2-bromorthanol as initiator and Sn(Oct)2 as catalyst according to literature [13]. Typically, ε-CL (10 g), 2-bromorthanol (0.2 g) and Sn(Oct)2 (0.034 g) were dissolved into 10 mL toluene in a round-bottom flask. After the cycles of freeze-pump-thaw to remove moisture and oxygen, the polymerization was performed in an oil bath at 120 ℃ for 24 h. The product was collected by precipitation in excess of cold diethyl ether. The obtained PCL-Br (4.0 g, 0.66 mmol) and 0.8 g (6.6 mmol) of BM were dissolved in 40.0 mL DMF. Then 1.16 mL (6.6 mmol) of DIEA was injected into the flask by a syringe under nitrogen atmosphere. The reaction mixture was stirred at 70 ℃ under nitrogen for 24 h. The solution was concentrated by rotary evaporator, and then redissolved into trichloromethane and washed with double-distilled water. The solution was dried with anhydrous magnesium sulfate (MgSO4) and precipitated in cold diethyl ether. The product was then obtained through drying the precipitate under vacuum at 30 ℃. The final yield of BM-PCL was 73.22 %. 2.3. Preparation of CS-g-CD/BM-PCL complex micelles 6
The CS-g-CD/BM-PCL complex micelles were formed via host-guest interactions between β-CD and BM. Briefly, CS-g-CD was dissolved in amount of 1.0 % acetic acid aqueous solution with a concentration of 0.1 mg/mL. Subsequently, the solution was then neutralized to pH 7.0 by NaOH. After that, BM-PCL was dissolved in DMF with a concentration of 0.1 mg/mL. Different proportion of BM-PCL solution was added dropwise to CS-g-CD solution with magnetic stirring. The mixed solutions were kept stirring for 24 h at room temperature to ensure full inclusion complexation. 2.4. Drug-loaded in CS-g-CD/BM-PCL complex micelles The drug-loaded polymer micelles were prepared by using DOX as a model drug at 25 ℃. Typically, a certain amount of DOX was dissolved in CS-g-CD solution with a concentration of 0.1 mg/mL, and then isoconcentration of BM-PCL solution was added dropwise into the above solution under stirring. After incubation for 24 h, the solution was transferred into a dialysis membrane bag (MWCO 14 kDa, Biosharp) subjected to dialysis against distilled water to remove non-encapsulated drugs. The concentration of DOX in dialysate fluid was analyzed by UV absorbance at the wavelength of 495 nm. Each sample was measured in triplicate. The amount of drug in micelles was calculated by subtracting the amount of drug from the dialysate fluid. The drug-loading content (DLC %) and entrapment efficiency (EE %) of complex micelles were calculated according to the following equations [28]. DLC%
m0 cV 100% m m0 cV
(1)
7
EE %
m0 cV 100% m0
(2)
Where m0 is drug dosage; m is the weight of hollow microspheres; c is the concentration of DOX in dialysis fluid; V is the volume of dialysis solutions. 2.5. Drug release from CS-g-CD/BM-PCL complex micelles In vitro release profiles of DOX from drug-loaded micelles were studied using a UV-vis spectrophotometer. After dialysis, 8.0 mL of DOX-loaded micelles were transferred into a dialysis pocket, and then placed into 50 mL different phosphate buffer solution in a thermostatic water bath. The drug release was studied at a given temperature (25 ℃, 37 ℃) and pH (2.0, 5.2, 7.0). At predetermined time intervals, 5.0 mL of external release medium was taken out for analysis and an equal volume of fresh release medium was added simultaneously into the release system. The concentration of released DOX was determined by UV spectrophotometer at 495 nm. The cumulative drug release was calculated according to formula (3). The analysis was performed in triplicate for each sample and the results were the average of three runs. Cumulative release
50Cn 5.0 Cn 1
(3)
m0 cV
Where Cn and Cn-1 are the concentration of DOX released from the drug delivery system at n and n-1 times, respectively; n is the time of drawing out the buffer solution(n＞0); other symbols are as the same above mentioned. 2.6. Cytotoxicity studies The cytotoxicity of drug-loaded vesicles was examined via MTT assay. Hela cells 8
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) in 5 % CO2. The cells were seeded in a 96 well plate with a density of 5000 cells per well and incubated for 24 h. The vesicles solution was diluted with culture medium to achieve different concentrations. 100 μL of the sample solution was used to replace the medium in each well, and the plates were incubated at 37 ℃ in 5 % CO2 for 48 h. The culture medium and 10 μL MTT were used to replace the mixture in each well and incubated for 4 h. The media were completely removed and 150 μL DMSO was added to dissolve the formazane crystals formed by viable cells. The optical density of the solution was measured using a microplate reader at 570 nm. 2.7. Characterizations Structure analysis of polymers and complex micelles were carried out with a fourier transform infrared (FTIR) spectroscopy (MAGNA550, Nicolet, USA). The complex micelles solution was frozen at -18 ℃ and lyophilized by a freeze-dryer to obtain dried powder. 1HNMR spectra were recorded on a Bruker AV 300 NMR spectrometer in chloroform. Elemental analysis was conducted by the Elementar vario EL cube (Elementar Corporation, Germany). The X-ray diffraction (XRD) experiments were performed by the transfer target X-ray polycrystalline diffraction (Rigakud/Max2500, electronics corporation, Japan). The transmittance measurement of complex micelles solutions was carried out using a UV-Vis spectrophotometer (UV-2550, Shimadzu, Japan). Morphological evaluation of complex micelles was performed with a transmission electron microscope (TEM, JEM-1230, JEOL, Japan) operating at an acceleration voltage of 200 kV. The size distribution of micelles was 9
performed by laser particle analyzer experiments using a Nano ZS90 laser particle analyzer (Malvern) at a scattering angle of 90o. 3. Results and discussion 3.1. Characterization of CS-g-CD and BM-PCL FTIR was used to confirm the chemical structure of synthesized copolymers. As shown in Fig. 1a, the spectrum of β-CD showed absorption band at wavenumber of 947 cm−1, which is the characteristic absorption peak of α-pyran from cyclodextrins. The absorption band at 1156 cm−1 was corresponding to the symmetric stretching of C-O-C bridge, while the peaks at 1079 cm−1 and 1030 cm−1 were the skeletal vibration of C-O-C involving C-O stretching [29,30]. In Fig. 1b, the additional absorption at wavenumbers of 1597 cm−1, 839 cm−1 and 815 cm−1 were the characterized absorption peaks of benzene ring of 6-OTs-β-CD. In Fig. 1c, the FTIR spectrum of CS showed two strong peaks at 3430 cm−1 and 1630 cm−1 due to -OH stretching vibration and -NH bending vibration, respectively. On comparing with the spectrum of CS, the appearance of new peak at 1028 cm−1 in CS-g-CD was due to the involved skeletal vibration of C-O stretching. The new peak at 945 cm−1 was the characteristic peak of α-pyran vibration from β-CD. These results confirmed that that CS-g-CD was successfully synthesized. The 1H NMR spectra of PCL-Br and BM-PCL in CDCl3 were shown in Fig. 2. In the 1H NMR spectrum of PCL-Br, the peaks at δ=2.46, 1.81, 1.54, 1.42 and 4.06 ppm were belonged to the methylene protons in the main chain of PCL. In the spectrum of BM-PCL, the peaks at 3.4 and 4.4 ppm disappeared, which were assigned to the 10
methylene protons close to bromine. The peaks at 7.3 and 7.8 ppm belonged to benzene protons and the peak at 8.4 ppm belonged to imidazole proton were all appeared in BM-PCL. These results indicated the benzimidazole group was conjugated onto PCL [13]. Furthermore, the elemental analysis showed that the content of N element in BM-PCL was 1.18%. The result also proved the successful synthesis of BM-PCL. The number-average molecule weight (Mn) of BM-PCL measured from GPC was 6600 g/mol. 3.2. Characterization of CS-g-CD/BM-PCL complex micelles In neutral solutions, the hydrophobic benzimidazole group in BM-PCL can insert in the cavity of β-CD from CS-g-CD by host-guest interactions to yield complex micelles. In order to prove the host-guest molecular interactions, the FTIR spectra of CS-g-CD, BM-PCL and their complexes were characterized as shown in Fig. 3. The characteristic peak at 942 cm-1 was assigned to the sugar ring skeleton vibration absorption peaks from cyclodextrins in CS-g-CD, which was also observed in complex micelles. The peak at 1361 cm-1 aroused from tertiary amine of benzimidazole group from BM-PCL decreased or disappeared in the spectrum of complex micelles [31]. The carbonyl stretching vibration of PCL in complex micelles shifted from 1726 cm-1 to 1733 cm-1 and became weaker due to the effect of complexation. These results suggested that guest molecule BM-PCL formed targeted complexes with CS-g-CD through interactions. The formation of inclusion complexes via host-guest interactions between CS-g-CD and BM-PCL was further verified by XRD as shown in Fig. 4. BM-PCL 11
showed the characteristic peaks at 21.5° and 23.6° in XRD spectrum. While the characteristic peaks aroused from BM-PCL almost disappeared in complex micelles. There was only one broad peak at 21.2° in the spectrum of complex micelles. The decrease of crystallinity for complex micelles is because the PCL chain was bound in the cavity of cyclodextrin. The low crystallinity indicated that the complex micelles were formed [32,33]. Formation of assemblies from CS-g-CD and BM-PCL was also confirmed by the changes of transmittance for mixture solutions. CS-g-CD was dissolved in acetic acid aqueous solution. With adding designed volume of BM-PCL, inclusion complexation between CS-g-CD and BM-PCL occurred resulting in the formation of assemblies. The inclusion complexation was accompanied by the decrease of transmittance as shown in Fig. 5. With the increase of BM-PCL, the transmittance of mixtures first decreased and then increased. When the ratio of CS-g-CD to BM-PCL was 3:1 (v/v), the light transmittance of complex micelles reached to the lowest and the solution was homogeneous. At this condition, stable complex micelles were formed between CS-g-CD and BM-PCL. In following discussions, we used this ratio as the research sample for other characterizations. 3.3. Morphology and size analysis of CS-g-CD/BM-PCL complex micelles The morphology of CS-g-CD/BM-PCL complex micelles, drug loaded complex micelles and micelles after release was observed by TEM as shown in Fig. 6. It was clearly that the blank micelles and the drug loaded micelles were observed as regular spherical structure with good dispersity. Obviously, the micelles were formed through 12
host-guest interactions with β-CD/BM-PCL as the core, which was stabilized by the hydrophilic chitosan chains as the shell. The size of the blank micelles without drug was in a range of 50-00 nm, while the size of micelles after drug loaded increased to 100-150 nm. After drug loading, the complex micelles still keep good shape as spheres, which is in favour of the transportation of drugs in the circulation. But the micelles dissociated into small aggregrates or fragment after drug release as shown in Fig. 6C. Fig. 7 shows the hydrodynamic diameter distributions f(Dh) of complex micelles measured by laser particle analyzer at room temperature. As shown in Fig. 7, the average hydrodynamic diameter of CS-g-CD/BM-PCL complex micelles was determined about 200 nm with narrow size distribution. The size of micelles was slightly larger than that of observed by TEM. This is because the micelles were swollen in water, while TEM observation showed the diameter of dried micelles. 3.4. Encapsulation of DOX in CS-g-CD/BM-PCL complex micelles DOX is one of the effective chemotherapeutic anticancer drugs, but it has short biological half-life in vivo and seriously hurts the healthy cells when it kills the tumor cells [34]. To improve its bioavailability, reduce the side effects and increase the therapeutic efficacy, stimuli-responsive micelles have been used as carriers for DOX [35]. In this study, DOX was selected as a model drug to determine the encapsulation and release behavior of complex micelles. The drug-loading content (DLC %) and entrapment efficiency (EE %) of CS-g-CD/BM-PCL complex micelles were calculated as shown in Table 1. It was shown that the DLC % of complex micelles 13
increased from 11.64% to 36.38% with the increase of drug dosage. The EE % changed a little and reached up to 74.88 % when the drug dosage was 60%. Compared with other nanocarriers from CS-g-CD and sodium tripolyphosphate reported in our previous work [36], the drug loading content and entrapment efficiency of CS-g-CD/BM-PCL complex micelles were larger. The high loading capacity of micelles was because that DOX could be effectively loaded into the micelles core via hydrophobic interactions, which is composed of PCL and β-CD. What is more important, DOX could be wrapped into the hydrophobic inner cavity of β-CD. The two reasons were contributed to the improvement of drug loading. Therefore, complex micelles assembled from CS-g-CD and BM-PCL can offer promising features in drug delivery. In the following experiment, the dosage ratio of 60 % was chosen to measure the drug release. 3.5. Release of DOX from CS-g-CD/BM-PCL complex micelles The in vitro release behavior of DOX from CS-g-CD/BM-PCL complex micelles was investigated at different temperature and pH. Fig. 8 shows the cumulative release of DOX at different pH buffer solutions at 37 ℃. The DOX release profiles exhibit two distinct stages. In the first stage, a faster drug release occurred within 10 h, followed by a sustained drug-release period, attained over a prolonged time. The initial fast release from polymeric micelles may be attributed to the remaining DOX on the micelles surface. The cumulative release of DOX from complex micelles increased with the decrease of pH values. Only about 56 % of DOX was released at pH 7.0 while 85 % of DOX was released at pH 5.2. The pH-dependent release of 14
DOX is attributed to the acid-triggered disassembly of micelles at acid condition. At neutral solution, release of DOX from micelles was suppressed because the micelles were stable at this condition due to the complexation of CS-g-CD with BM-PCL. At weak acid condition (pH 5.2), the protonation of BM group resulted in the dissociation of complexes between BM and β-CD. As a result, the fast drug release was realized. At pH 2.0, the cumulative release was close to 100% within 10 h. The other reason is might that β-CD was unstable at strong acid conditions resulting in the release of hydrophobic drugs from interior cavity of cyclodextrins. The accelerated drug release in acidic environment is preferred in a practical drug delivery system for anticancer. Fig. 9 shows the cumulative release of CS-g-CD/BM-PCL complex micelles at different temperatures. It revealed that the temperature also had significant effect on the drug release. Only about 60 % of DOX was released into surroundings at 25 ℃, while 100 % of DOX was released at 37 ℃. This is mainly because the micelles were stable at room temperature due to the host-guest interactions between BM group and β-CD. The hydrogen bonding and Van der Waals interactions hindered the drug release from micelles [13].With the increase of temperatures, the interactions between BM and β-CD as well as the interactions between drugs and polymers would be weakened, which was beneficial for the drug release [37]. Therefore, such stimuli-responsive complex micelles are expected to be applied in drug delivery system in response to environmental stimuli. 3.6. Cytotoxicity evaluations 15
To examine the cytotoxicity of CS-g-CD/BM-PCL complex micelles, Hela cells were exposed to various doses of blank micelles and drug-loaded micelles for 48 h. From Fig. 10, it was seen that the cell viabilities for blank micelles remained over 100 % at concentrations up to 0.5 mg/mL. This demonstrated that no apparent cytotoxicity for blank micelles even at higher concentrations. However, the cell viability of drug-loaded micelles declined remarkably with the increase of concentration. It was obvious that lower cell viability was achieved for drug carriers by increasing the concentration of drug-loaded particles. 4. Conclusion In summary, sensitive complex micelles prepared from CS-g-CD and BM-PCL through host-guest recognition were studied. The results suggested that the inclusion complexation interactions between β-cyclodextrin and benzimidazole resulted in the formation of aggregates. The micelles, with an average diameter of about 200 nm, presented high encapsulation efficiency for DOX. The release of DOX from complex micelles was enhanced with the pH decreased from 7.0 to 2.0 and the temperature increased from 25 to 37 ℃. Cytotoxicity assays revealed that these polymer micelles exhibited good biocompatibility, while drug-loaded micelles retained higher cell inhibition efficiency. These sensitive complex micelles may serve as potential smart drug delivery used in hydrophobic-drug delivery and controlled release vehicle. Acknowledgment The authors thank for the financial support from National Natural Science Foundation of China (21204035 and 51403096) and Shandong Province Higher 16
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[27] R. Petter, J. Salek, C. Sikorski, G. Kumaravel, F. Lin, Cooperative binding by aggregated mono-6-(alkylamino)-beta-cyclodextrins, J. Am. Chem. Soc. 112 (1989) 3860-3868. [28] G. Li, M. Qi, N. Yu, X. Liu, Hybrid vesicles co-assembled from anionic graft copolymer and metal ions for controlled drug release, Chem. Eng. J. 262 (2015) 710-715. [29] A. Zhu, T. Chen, L. Yuan, H. Wu, P. Lu, Synthesis and characterization of N-succinyl-chitosan and its self-assembly of nanospheres, Carbohyd. Polym. 66(2006) 274-279. [30] P. Gonil, W. Sajomsang, U. Ruktanonchai, N. Pimpha, I. Sramala, O. Nuchuchua, S. Saesoo, S.
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Puttipipatkhachorn,
Novel
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[35] W. Xu, I. Siddiqui, M. Nihal, S. Pilla, K. Rosenthal, H. Mukhtar, S. Gong, Aptamer-conjugated and doxorubicin-loaded unimolecular micelles for targeted therapy of prostate cancer, Biomaterials. 34 (2013) 5244-5253. [36] N. Yu, G. Li, Y. Gao, X. Liu, S. Ma, Stimuli-sensitive hollow spheres from chitosan-graft-β-cyclodextrin for controlled drug release, Int. J. Biol. Macromol. 93 (2016) 971-977. [37] V. Jain, P. Bharatam, Pharmacoinformatic approaches to understand complexation of dendrimeric nanoparticles with drugs, Nanoscale. 6 (2014) 2476-2501.
21
Figure Captions: Scheme 1. Formation of complex micelles from CS-g-CD and BM-PCL. Fig. 1. FTIR spectra of (a) β-CD, (b) 6-OTs-CD, (c) CS and (d) CS-g-CD. Fig. 2. 1H NMR spectra of (a) PCL-Br and (b) BM-PCL in CDCl3. Fig. 3. FTIR spectra of (a) CS-g-CD, (b) BM-PCL and (c) CS-g-CD/BM-PCL. Fig. 4. X-ray diffraction patterns of (a) BM-PCL, (b) CS-g-CD and (c) CS-g-CD/BM-PCL micelles. Fig. 5. The transmittance of CS-g-CD/BM-PCL complex micelles at different ratio. Fig. 6. TEM images of CS-g-CD/BM-PCL complex micelles (A) before drug loading, (B) after drug loading, and (C) after drug release. Fig. 7. Hydrodynamic diameters of CS-g-CD/BM-PCL complex micelles at 25 ℃. Fig. 8. Release of DOX from CS-g-CD/BM-PCL complex micelles at different pH (T = 37 ℃). Fig. 9. Release of DOX from CS-g-CD/BM-PCL complex micelles at different temperature (pH = 5.2). Fig. 10. Cytotoxicity of CS-g-CD/BM-PCL blank micelles and drug-loaded micelles against Hela cells.
22
23
24
25
26
27
28
Table 1. Drug-loading content (DLC%) and entrapment efficiency (EE%) of CS-g-CD/BM-PCL complex micelles for DOX. m0/m
DLC (%)
EE (%)
20%
11.64
65.85
40%
21.78
69.63
60%
30.97
74.77
80%
36.38
71.50
m0 is the weight of drug dosage; m is the weight of complex micelles.
29
S0141-8130(17)32368-1 http://dx.doi.org/doi:10.1016/j.ijbiomac.2017.06.120 BIOMAC 7800
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
26-2-2017 18-5-2017 29-6-2017
Please cite this article as: Yurong Gao, Guiying Li, Zaishuai Zhou, Lingling Gao, Qian Tao, Sensitive complex micelles based on host-guest recognition from chitosan-graft--cyclodextrin for drug release, International Journal of Biological Macromoleculeshttp://dx.doi.org/10.1016/j.ijbiomac.2017.06.120 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Sensitive complex micelles based on host-guest recognition from chitosan-graft-β-cyclodextrin for drug release Yurong Gao, Guiying Li, Zaishuai Zhou, Lingling Gao, Qian Tao School of Chemistry and Materials Science, Ludong University, Yantai 264025, China
Corresponding author.
E-mail address: [email protected] (G. Li).
Abstract: In this paper, pH-sensitive complex micelles were developed based on the host-guest
recognition
from
chitosan-graft-β-cyclodextrin
(CS-g-CD)
and
benzimidazole-terminated poly(ε-caprolactone) (BM-PCL) for controlled drug release. The formation and characterization of complex micelles were confirmed by fourier-transform
infrared
spectroscopy
(FTIR),
X-ray
diffraction
(XRD),
transmission electron microscopy (TEM) and laser particle analyzer. The size of complex micelles was about 200 nm with the core formed by BM-PCL/β-CD and the shell composed of chitosan. Doxorubicin (DOX), a model anticancer drug, was effectively loaded into the complex micelles via hydrophobic interactions. The encapsulation efficiency of DOX was up to 75 %. The release of DOX from complex micelles was suppressed at neutral pH solutions due to the stability of micelles but accelerated at acidic solutions and high temperatures. These sensitive complex micelles might possess potential applications as intelligent nanocarriers for anticancer drug delivery. Key words: Benzimidazole, Chitosan-graft-β-cyclodextrin, Controlled release,
1
Host-guest recognition, Sensitive polymer micelles 1. Introduction In recent years, nanoparticles have been received extensive investigations due to their potential applications in diverse areas, such as gene and drug delivery, biological sensors, hydrogen storage materials, and so on [1-3]. Among these nanoparticles, polymer micelles aggregated from amphiphilic copolymers are characterized by core-shell structure with the core formed by hydrophobic blocks and the shell formed by hydrophilic blocks. The micelles have been emerged as potential nanocarriers which the inner core can be used to encapsulate hydrophobic drugs and the outer shell offer attractive characteristics such as suitable size, low toxicity for human body, etc [4-7]. Compared with conventional polymer micelles, environmentally sensitive polymer micelles, which show intelligent response to environmental changes, such as pH, temperature, ionic strength, and so on, are most intensively studied in drug delivery systems. Sensitive polymer micelles have distinct properties including high encapsulation efficiency and drug loading, stimuli-responsive property and controlled release character, etc [8-10]. Owing to the fact that different part in human body has different pH environments, pH-sensitive polymer micelles are the best candidate for preparation of drug carriers. The pH of human body is maintained at about 7.4 in normal extracellular matrices and blood, while the solid tumors always present as acidic condition (pH ~6.0) due to the inefficient consumption of glucose into energy in cancer cells producing a large amount of lactic acid [11,12]. Therefore, pH-sensitive polymer nanocarriers have been developed for tumor targeting and 2
intracellular drug release [13]. Cyclodextrins (CDs), a series of natural macrocyclic oligosaccharides with a hydrophilic exterior and a hydrophobic interior cavity, have been extensively used in pharmaceutical field [14-17]. The hydrophobic internal cavity of CDs gives them inclusion capacity with large quantities of guest molecules, such as various poorly water-soluble drugs. Therefore, CDs have been utilized to encapsulate drugs through host-guest interactions in drug delivery system [18,19]. Such encapsulation may immensely increase the solubility of lipophilic drugs, improve the stability of labile drugs, alleviate the local and systemic toxicity, and control the drug release profiles [20-22]. For example, Tu et al. synthesized supramolecular star-shaped ABC terpolymer via the molecular recognition between β-CD based diblock copolymer poly(dimethylaminoethyl modified
methyl
pH-responsive
methacrylate)-poly(ethylene
methacrylate supramolecular
benzimidazole-terminated
(AD-PMMA)
[23].
amphiphilic
poly(ethylene
glycol)
and
Zhang et micelles
glycol)
adamantane al.
reported
based
(BM-PEG)
on and
β-cyclodextrin-modified poly(L-lactide) (CD-PLLA) by using host-guest interactions between benzimidazole and β-cyclodextrin [24]. It is well known that the host-guest interactions between β-cyclodextrin (β-CD) and benzimidazole (BM) exhibit pH-sensitivity [25]. Under the neutral conditions, the BM molecule is hydrophobic, which can be bound to β-CD cavity by host-guest interactions. The nitrogen atoms of BM are protonated under acidic conditions, which result in the dissociation of complexation between β-CD and BM [13,26]. By 3
adjusting the pH of solutions, the assembly-disassembly between BM and β-CD can be controlled. Chitosan (CS), which shows several biological properties, such as extremely nontoxicity, biodegradability, and excellent biocompatibility, is also well known for pH sensitive polymers due to the existence of -NH2 groups [10]. Combining the advantages of cyclodextrin with chitosan is conducive to improve the drug loading and pH-sensitivity of nanocarriers. Based on this conception, we designed sensitive polymer complex micelles due to the host-guest recognitions between chitosan graft β-cyclodextrin (CS-g-CD) and benzimidazole-terminated poly(ε-caprolactone) (BM-PCL). The micelles had a core-shell structure with the core formed from BM-PCL and β-CD via host-guest interactions and the shell composed of chitosan chains as shown in Scheme 1. The structure, morphology and particle size of micelles were analyzed using fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM) and laser particle analyzer. Doxorubicin (DOX), an anthracycline anticancer drug, was chosen as a model drug to access the applications of CS-g-CD/BM-PCL complex micelles in drug delivery. 2. Experimental 2.1. Materials Chitosan (CS) (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China) with a deacetylation degree of 95 % and a viscosity-average molecular weight of 2.0 × 105 g/mol was purified by solubilization in aqueous acetic acid and reprecipitation by aqueous NaOH. β-Cyclodextrin (Shanghai Chemical Reagent Company, China) was 4
recrystallized twice from water and dried in vacuum at 80 ℃. p-Toluenesulfonyl chloride was provided from Damao Chemical Reagent Co. Ltd. (Tianjin, China). 2-Bromorthanol, ε-caprolactone (ε-CL), benzimidazole (BM), stannous octoate (Sn(Oct)2) and N,N-diisopropylethylamine (DIEA) were purchased from Shanghai Macklin
Biochemical
Co.
Ltd.
(China).
Doxorubicin
(DOX)
and
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylterazolium bromide (MTT) were purchased from J&K Chemical Ltd. (Beijing, China). All other reagents used were commercially available and were of analytical grade. Double-distilled water was used for the preparation of all solutions in this study. 2.2. Preparation of copolymers 2.2.1. Preparation of chitosan-graft-β-cyclodextrin (CS-g-CD) CS-g-CD
was
synthesized
from
CS
and
mono-6-deoxy-6-(p-toluenesulfonyl)-β-cyclodextrin (6-OTs-β-CD) as described in previous reports [16,27]. Briefly, β-CD (15.0 g, 13.2 mmol) was suspended in 125.0 mL of distilled water, and then NaOH solution (5 mL, 41.1 mmol) was slowly dripped into the solution. p-Toluenesulfonyl chloride (2.5 g, 13.2 mmol) in 7.5 mL of acetonitrile was cautiously dripped into the above solution, causing immediate formation of a white sediment. After 3 h of stirring at 5 ℃, the filtrate was collected by filtration. The pH of solution was adjusted to 6.0 by hydrochloric acid, and then the solution was placed in the refrigerator overnight at 4 ℃. The precipitation of 6-OTs-β-CD was recrystallized twice from water and dried in vacuum at 50 ℃. Then, CS (0.5 g) was dissolved in 1.0 % acetic acid aqueous solution (pH 3.0, 40 mL). The 5
6-OTs-β-CD (0.5 g) was dissolved in 20 mL N,N-dimethylformamide (DMF) before it was added into CS solution. The reaction mixture was refluxed at 100 ℃ for 14 h after purging with nitrogen. The raw products CS-g-CD were purified by soxhlets using acetone for 48 h and then dried in vacuum. The yield of CS-g-CD was calculated to be 65.58 % by the gravimetric analysis. 2.2.2. Preparation of BM-PCL PCL-Br was synthesized by ring-opening polymerization of ε-caprolactone monomer using 2-bromorthanol as initiator and Sn(Oct)2 as catalyst according to literature [13]. Typically, ε-CL (10 g), 2-bromorthanol (0.2 g) and Sn(Oct)2 (0.034 g) were dissolved into 10 mL toluene in a round-bottom flask. After the cycles of freeze-pump-thaw to remove moisture and oxygen, the polymerization was performed in an oil bath at 120 ℃ for 24 h. The product was collected by precipitation in excess of cold diethyl ether. The obtained PCL-Br (4.0 g, 0.66 mmol) and 0.8 g (6.6 mmol) of BM were dissolved in 40.0 mL DMF. Then 1.16 mL (6.6 mmol) of DIEA was injected into the flask by a syringe under nitrogen atmosphere. The reaction mixture was stirred at 70 ℃ under nitrogen for 24 h. The solution was concentrated by rotary evaporator, and then redissolved into trichloromethane and washed with double-distilled water. The solution was dried with anhydrous magnesium sulfate (MgSO4) and precipitated in cold diethyl ether. The product was then obtained through drying the precipitate under vacuum at 30 ℃. The final yield of BM-PCL was 73.22 %. 2.3. Preparation of CS-g-CD/BM-PCL complex micelles 6
The CS-g-CD/BM-PCL complex micelles were formed via host-guest interactions between β-CD and BM. Briefly, CS-g-CD was dissolved in amount of 1.0 % acetic acid aqueous solution with a concentration of 0.1 mg/mL. Subsequently, the solution was then neutralized to pH 7.0 by NaOH. After that, BM-PCL was dissolved in DMF with a concentration of 0.1 mg/mL. Different proportion of BM-PCL solution was added dropwise to CS-g-CD solution with magnetic stirring. The mixed solutions were kept stirring for 24 h at room temperature to ensure full inclusion complexation. 2.4. Drug-loaded in CS-g-CD/BM-PCL complex micelles The drug-loaded polymer micelles were prepared by using DOX as a model drug at 25 ℃. Typically, a certain amount of DOX was dissolved in CS-g-CD solution with a concentration of 0.1 mg/mL, and then isoconcentration of BM-PCL solution was added dropwise into the above solution under stirring. After incubation for 24 h, the solution was transferred into a dialysis membrane bag (MWCO 14 kDa, Biosharp) subjected to dialysis against distilled water to remove non-encapsulated drugs. The concentration of DOX in dialysate fluid was analyzed by UV absorbance at the wavelength of 495 nm. Each sample was measured in triplicate. The amount of drug in micelles was calculated by subtracting the amount of drug from the dialysate fluid. The drug-loading content (DLC %) and entrapment efficiency (EE %) of complex micelles were calculated according to the following equations [28]. DLC%
m0 cV 100% m m0 cV
(1)
7
EE %
m0 cV 100% m0
(2)
Where m0 is drug dosage; m is the weight of hollow microspheres; c is the concentration of DOX in dialysis fluid; V is the volume of dialysis solutions. 2.5. Drug release from CS-g-CD/BM-PCL complex micelles In vitro release profiles of DOX from drug-loaded micelles were studied using a UV-vis spectrophotometer. After dialysis, 8.0 mL of DOX-loaded micelles were transferred into a dialysis pocket, and then placed into 50 mL different phosphate buffer solution in a thermostatic water bath. The drug release was studied at a given temperature (25 ℃, 37 ℃) and pH (2.0, 5.2, 7.0). At predetermined time intervals, 5.0 mL of external release medium was taken out for analysis and an equal volume of fresh release medium was added simultaneously into the release system. The concentration of released DOX was determined by UV spectrophotometer at 495 nm. The cumulative drug release was calculated according to formula (3). The analysis was performed in triplicate for each sample and the results were the average of three runs. Cumulative release
50Cn 5.0 Cn 1
(3)
m0 cV
Where Cn and Cn-1 are the concentration of DOX released from the drug delivery system at n and n-1 times, respectively; n is the time of drawing out the buffer solution(n＞0); other symbols are as the same above mentioned. 2.6. Cytotoxicity studies The cytotoxicity of drug-loaded vesicles was examined via MTT assay. Hela cells 8
were cultured in Dulbecco’s modified Eagle’s medium (DMEM) in 5 % CO2. The cells were seeded in a 96 well plate with a density of 5000 cells per well and incubated for 24 h. The vesicles solution was diluted with culture medium to achieve different concentrations. 100 μL of the sample solution was used to replace the medium in each well, and the plates were incubated at 37 ℃ in 5 % CO2 for 48 h. The culture medium and 10 μL MTT were used to replace the mixture in each well and incubated for 4 h. The media were completely removed and 150 μL DMSO was added to dissolve the formazane crystals formed by viable cells. The optical density of the solution was measured using a microplate reader at 570 nm. 2.7. Characterizations Structure analysis of polymers and complex micelles were carried out with a fourier transform infrared (FTIR) spectroscopy (MAGNA550, Nicolet, USA). The complex micelles solution was frozen at -18 ℃ and lyophilized by a freeze-dryer to obtain dried powder. 1HNMR spectra were recorded on a Bruker AV 300 NMR spectrometer in chloroform. Elemental analysis was conducted by the Elementar vario EL cube (Elementar Corporation, Germany). The X-ray diffraction (XRD) experiments were performed by the transfer target X-ray polycrystalline diffraction (Rigakud/Max2500, electronics corporation, Japan). The transmittance measurement of complex micelles solutions was carried out using a UV-Vis spectrophotometer (UV-2550, Shimadzu, Japan). Morphological evaluation of complex micelles was performed with a transmission electron microscope (TEM, JEM-1230, JEOL, Japan) operating at an acceleration voltage of 200 kV. The size distribution of micelles was 9
performed by laser particle analyzer experiments using a Nano ZS90 laser particle analyzer (Malvern) at a scattering angle of 90o. 3. Results and discussion 3.1. Characterization of CS-g-CD and BM-PCL FTIR was used to confirm the chemical structure of synthesized copolymers. As shown in Fig. 1a, the spectrum of β-CD showed absorption band at wavenumber of 947 cm−1, which is the characteristic absorption peak of α-pyran from cyclodextrins. The absorption band at 1156 cm−1 was corresponding to the symmetric stretching of C-O-C bridge, while the peaks at 1079 cm−1 and 1030 cm−1 were the skeletal vibration of C-O-C involving C-O stretching [29,30]. In Fig. 1b, the additional absorption at wavenumbers of 1597 cm−1, 839 cm−1 and 815 cm−1 were the characterized absorption peaks of benzene ring of 6-OTs-β-CD. In Fig. 1c, the FTIR spectrum of CS showed two strong peaks at 3430 cm−1 and 1630 cm−1 due to -OH stretching vibration and -NH bending vibration, respectively. On comparing with the spectrum of CS, the appearance of new peak at 1028 cm−1 in CS-g-CD was due to the involved skeletal vibration of C-O stretching. The new peak at 945 cm−1 was the characteristic peak of α-pyran vibration from β-CD. These results confirmed that that CS-g-CD was successfully synthesized. The 1H NMR spectra of PCL-Br and BM-PCL in CDCl3 were shown in Fig. 2. In the 1H NMR spectrum of PCL-Br, the peaks at δ=2.46, 1.81, 1.54, 1.42 and 4.06 ppm were belonged to the methylene protons in the main chain of PCL. In the spectrum of BM-PCL, the peaks at 3.4 and 4.4 ppm disappeared, which were assigned to the 10
methylene protons close to bromine. The peaks at 7.3 and 7.8 ppm belonged to benzene protons and the peak at 8.4 ppm belonged to imidazole proton were all appeared in BM-PCL. These results indicated the benzimidazole group was conjugated onto PCL [13]. Furthermore, the elemental analysis showed that the content of N element in BM-PCL was 1.18%. The result also proved the successful synthesis of BM-PCL. The number-average molecule weight (Mn) of BM-PCL measured from GPC was 6600 g/mol. 3.2. Characterization of CS-g-CD/BM-PCL complex micelles In neutral solutions, the hydrophobic benzimidazole group in BM-PCL can insert in the cavity of β-CD from CS-g-CD by host-guest interactions to yield complex micelles. In order to prove the host-guest molecular interactions, the FTIR spectra of CS-g-CD, BM-PCL and their complexes were characterized as shown in Fig. 3. The characteristic peak at 942 cm-1 was assigned to the sugar ring skeleton vibration absorption peaks from cyclodextrins in CS-g-CD, which was also observed in complex micelles. The peak at 1361 cm-1 aroused from tertiary amine of benzimidazole group from BM-PCL decreased or disappeared in the spectrum of complex micelles [31]. The carbonyl stretching vibration of PCL in complex micelles shifted from 1726 cm-1 to 1733 cm-1 and became weaker due to the effect of complexation. These results suggested that guest molecule BM-PCL formed targeted complexes with CS-g-CD through interactions. The formation of inclusion complexes via host-guest interactions between CS-g-CD and BM-PCL was further verified by XRD as shown in Fig. 4. BM-PCL 11
showed the characteristic peaks at 21.5° and 23.6° in XRD spectrum. While the characteristic peaks aroused from BM-PCL almost disappeared in complex micelles. There was only one broad peak at 21.2° in the spectrum of complex micelles. The decrease of crystallinity for complex micelles is because the PCL chain was bound in the cavity of cyclodextrin. The low crystallinity indicated that the complex micelles were formed [32,33]. Formation of assemblies from CS-g-CD and BM-PCL was also confirmed by the changes of transmittance for mixture solutions. CS-g-CD was dissolved in acetic acid aqueous solution. With adding designed volume of BM-PCL, inclusion complexation between CS-g-CD and BM-PCL occurred resulting in the formation of assemblies. The inclusion complexation was accompanied by the decrease of transmittance as shown in Fig. 5. With the increase of BM-PCL, the transmittance of mixtures first decreased and then increased. When the ratio of CS-g-CD to BM-PCL was 3:1 (v/v), the light transmittance of complex micelles reached to the lowest and the solution was homogeneous. At this condition, stable complex micelles were formed between CS-g-CD and BM-PCL. In following discussions, we used this ratio as the research sample for other characterizations. 3.3. Morphology and size analysis of CS-g-CD/BM-PCL complex micelles The morphology of CS-g-CD/BM-PCL complex micelles, drug loaded complex micelles and micelles after release was observed by TEM as shown in Fig. 6. It was clearly that the blank micelles and the drug loaded micelles were observed as regular spherical structure with good dispersity. Obviously, the micelles were formed through 12
host-guest interactions with β-CD/BM-PCL as the core, which was stabilized by the hydrophilic chitosan chains as the shell. The size of the blank micelles without drug was in a range of 50-00 nm, while the size of micelles after drug loaded increased to 100-150 nm. After drug loading, the complex micelles still keep good shape as spheres, which is in favour of the transportation of drugs in the circulation. But the micelles dissociated into small aggregrates or fragment after drug release as shown in Fig. 6C. Fig. 7 shows the hydrodynamic diameter distributions f(Dh) of complex micelles measured by laser particle analyzer at room temperature. As shown in Fig. 7, the average hydrodynamic diameter of CS-g-CD/BM-PCL complex micelles was determined about 200 nm with narrow size distribution. The size of micelles was slightly larger than that of observed by TEM. This is because the micelles were swollen in water, while TEM observation showed the diameter of dried micelles. 3.4. Encapsulation of DOX in CS-g-CD/BM-PCL complex micelles DOX is one of the effective chemotherapeutic anticancer drugs, but it has short biological half-life in vivo and seriously hurts the healthy cells when it kills the tumor cells [34]. To improve its bioavailability, reduce the side effects and increase the therapeutic efficacy, stimuli-responsive micelles have been used as carriers for DOX [35]. In this study, DOX was selected as a model drug to determine the encapsulation and release behavior of complex micelles. The drug-loading content (DLC %) and entrapment efficiency (EE %) of CS-g-CD/BM-PCL complex micelles were calculated as shown in Table 1. It was shown that the DLC % of complex micelles 13
increased from 11.64% to 36.38% with the increase of drug dosage. The EE % changed a little and reached up to 74.88 % when the drug dosage was 60%. Compared with other nanocarriers from CS-g-CD and sodium tripolyphosphate reported in our previous work [36], the drug loading content and entrapment efficiency of CS-g-CD/BM-PCL complex micelles were larger. The high loading capacity of micelles was because that DOX could be effectively loaded into the micelles core via hydrophobic interactions, which is composed of PCL and β-CD. What is more important, DOX could be wrapped into the hydrophobic inner cavity of β-CD. The two reasons were contributed to the improvement of drug loading. Therefore, complex micelles assembled from CS-g-CD and BM-PCL can offer promising features in drug delivery. In the following experiment, the dosage ratio of 60 % was chosen to measure the drug release. 3.5. Release of DOX from CS-g-CD/BM-PCL complex micelles The in vitro release behavior of DOX from CS-g-CD/BM-PCL complex micelles was investigated at different temperature and pH. Fig. 8 shows the cumulative release of DOX at different pH buffer solutions at 37 ℃. The DOX release profiles exhibit two distinct stages. In the first stage, a faster drug release occurred within 10 h, followed by a sustained drug-release period, attained over a prolonged time. The initial fast release from polymeric micelles may be attributed to the remaining DOX on the micelles surface. The cumulative release of DOX from complex micelles increased with the decrease of pH values. Only about 56 % of DOX was released at pH 7.0 while 85 % of DOX was released at pH 5.2. The pH-dependent release of 14
DOX is attributed to the acid-triggered disassembly of micelles at acid condition. At neutral solution, release of DOX from micelles was suppressed because the micelles were stable at this condition due to the complexation of CS-g-CD with BM-PCL. At weak acid condition (pH 5.2), the protonation of BM group resulted in the dissociation of complexes between BM and β-CD. As a result, the fast drug release was realized. At pH 2.0, the cumulative release was close to 100% within 10 h. The other reason is might that β-CD was unstable at strong acid conditions resulting in the release of hydrophobic drugs from interior cavity of cyclodextrins. The accelerated drug release in acidic environment is preferred in a practical drug delivery system for anticancer. Fig. 9 shows the cumulative release of CS-g-CD/BM-PCL complex micelles at different temperatures. It revealed that the temperature also had significant effect on the drug release. Only about 60 % of DOX was released into surroundings at 25 ℃, while 100 % of DOX was released at 37 ℃. This is mainly because the micelles were stable at room temperature due to the host-guest interactions between BM group and β-CD. The hydrogen bonding and Van der Waals interactions hindered the drug release from micelles [13].With the increase of temperatures, the interactions between BM and β-CD as well as the interactions between drugs and polymers would be weakened, which was beneficial for the drug release [37]. Therefore, such stimuli-responsive complex micelles are expected to be applied in drug delivery system in response to environmental stimuli. 3.6. Cytotoxicity evaluations 15
To examine the cytotoxicity of CS-g-CD/BM-PCL complex micelles, Hela cells were exposed to various doses of blank micelles and drug-loaded micelles for 48 h. From Fig. 10, it was seen that the cell viabilities for blank micelles remained over 100 % at concentrations up to 0.5 mg/mL. This demonstrated that no apparent cytotoxicity for blank micelles even at higher concentrations. However, the cell viability of drug-loaded micelles declined remarkably with the increase of concentration. It was obvious that lower cell viability was achieved for drug carriers by increasing the concentration of drug-loaded particles. 4. Conclusion In summary, sensitive complex micelles prepared from CS-g-CD and BM-PCL through host-guest recognition were studied. The results suggested that the inclusion complexation interactions between β-cyclodextrin and benzimidazole resulted in the formation of aggregates. The micelles, with an average diameter of about 200 nm, presented high encapsulation efficiency for DOX. The release of DOX from complex micelles was enhanced with the pH decreased from 7.0 to 2.0 and the temperature increased from 25 to 37 ℃. Cytotoxicity assays revealed that these polymer micelles exhibited good biocompatibility, while drug-loaded micelles retained higher cell inhibition efficiency. These sensitive complex micelles may serve as potential smart drug delivery used in hydrophobic-drug delivery and controlled release vehicle. Acknowledgment The authors thank for the financial support from National Natural Science Foundation of China (21204035 and 51403096) and Shandong Province Higher 16
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Figure Captions: Scheme 1. Formation of complex micelles from CS-g-CD and BM-PCL. Fig. 1. FTIR spectra of (a) β-CD, (b) 6-OTs-CD, (c) CS and (d) CS-g-CD. Fig. 2. 1H NMR spectra of (a) PCL-Br and (b) BM-PCL in CDCl3. Fig. 3. FTIR spectra of (a) CS-g-CD, (b) BM-PCL and (c) CS-g-CD/BM-PCL. Fig. 4. X-ray diffraction patterns of (a) BM-PCL, (b) CS-g-CD and (c) CS-g-CD/BM-PCL micelles. Fig. 5. The transmittance of CS-g-CD/BM-PCL complex micelles at different ratio. Fig. 6. TEM images of CS-g-CD/BM-PCL complex micelles (A) before drug loading, (B) after drug loading, and (C) after drug release. Fig. 7. Hydrodynamic diameters of CS-g-CD/BM-PCL complex micelles at 25 ℃. Fig. 8. Release of DOX from CS-g-CD/BM-PCL complex micelles at different pH (T = 37 ℃). Fig. 9. Release of DOX from CS-g-CD/BM-PCL complex micelles at different temperature (pH = 5.2). Fig. 10. Cytotoxicity of CS-g-CD/BM-PCL blank micelles and drug-loaded micelles against Hela cells.
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Table 1. Drug-loading content (DLC%) and entrapment efficiency (EE%) of CS-g-CD/BM-PCL complex micelles for DOX. m0/m
DLC (%)
EE (%)
20%
11.64
65.85
40%
21.78
69.63
60%
30.97
74.77
80%
36.38
71.50
m0 is the weight of drug dosage; m is the weight of complex micelles.
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