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Fabrication of Cellulose Nanocrystals-Based Folate Targeted Nanomedicine via Layer-by-Layer Assembly with Lysosomal pH-controlled Drug Release into Nucleus Na Li, Han Zhang, Yi Xiao, Yushu Huang, Mengda Xu, Donglei You, Wei Lu, and Jiahui Yu Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b01556 • Publication Date (Web): 08 Jan 2019 Downloaded from http://pubs.acs.org on January 9, 2019
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
FA/[email protected]@CNCs nanomedicines and illustration of FA mediated cellular uptake and efficient intracellular lysosomal pH controlled DOX release into nucleus was shown in Scheme 1.
Scheme 1. Schematic structure of pH-responsive FA/[email protected]@CNCs hybrids nanomedicnines and illustration of FA mediated endocytosis and efficient lysosomal pH controlled DOX release into nucleus.
CNCs was from Cellulose Lab (Canadian). Doxorubicin hydrochloride (DOX·HCl 98 %) was bought
Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl 99 %), N-hydroxysuccinimide (NHS), FA and PEI were bought from Sigma-Aldrich. Chemical agents (e.g. Cis-aconitic anhydride (CAA), dialysis bag and MTT (98 %), Hochest 33342) were got as we previously reported 26. Solvents (e.g. Ultrapure water, triethylamine (TEA) and so on) were prepared as we previously reported
Reagents not mentioned were used directly as received.
CAD was prepared as we previous report 26. FA/[email protected]@CNCs hybrids were prepared by the in-situ precipitate method via the LbL assembly. In detail, CNCs (10 mg) were dispersed into the PEI deionized water solution (25 mg/mL, 1mL), ultrasonication 15 min, 30 min later, the mix solution was added into the CAD solution (30 mg/mL, DMSO and water, 1mL) drop by drop, then, treated with centrifugation (14000 rpm,10 min) and washing (three times, deionized water). Finally, the deposition was dispersed into the FA solution (10 mg/mL, ethanol). After the treatments of centrifugation and washing, FA/[email protected]@CNCs nanomedicine was collected by lyophilization. 2.3 Characterization of physicochemical properties
-potentials of CNCs and FA/[email protected]@CNCs nanomedicine in aqueous phase were measured at 25 oC by dynamic light scattering (DLS) (Zetasizer Nano ZS, Malvern Instruments, UK). The concentration was maintained at 0.1 wt%. -potentials measurements were measured in automatic mode for an average of thirteen runs by Smoluchowski approximation in triplicates.
Thermogravimetric analyses (TGA)
TGA was performed using a Mettler Toledo Thermal Analysis Instruments (TGA/DSC 3+ United Kingdom). TGA balance was calibrated and the freeze-dried samples were analyzed in platinum pans under dry nitrogen purge at a flow rate of 50 ml/min from ambient temperature to 800 oC with a heating rate of 10 oC/min. The experimental conditions for data acquisition and analysis were performed on the STAR System.
Differential scanning calorimetry (DSC)
The thermal properties of CNCs and FA/[email protected]@CNCs nanomedicine were characterized by DSC using a differential scanning calorimeter (PerkinElmer, DSC 800, USA). CNCs and FA/[email protected]@CNCs were heated from − 40 to 200 oC under nitrogen atmosphere at a heating rate of 10 oC / min.
spectrophotometer (UV-vis) absorption spectra and a UV-1800 spectrometer were the same as reported 26.
The fluorescence intensity of DOX·HCl was measured by Fluorescence spectrum (FS). The
excitation and emission wavelength was 480 nm and 570 nm, respectively. The analysis operation were the same as reported 26 .
Atomic Force Microscopy (AFM) was used to study the morphology of CNCs and FA/[email protected]@CNCs on a Bruker Dimension Icon equipment with an integrated force generated by cantilever/silicon probes. One drop of a 0.01 wt% suspension of CNCs or CNCs based hybrids in water was deposited onto a freshly cleaved mica surface and allowed to dry in air under ambient conditions. Samples were scanned in contact mode in air.
X-ray powder diffractometer (XRD) with Cu Kα (1.5418 Å) radiation was measured on the FACS Calibur Ultima IV (Japan Science Co., Ltd. Japan). The angular of the diffractograms was set from 10º to 80º, and the scan speed was 0.1 s/step with a step size of 0.01º. The anode voltage and current was 50 kV and 40 mA, respectively. The Voigt profile shaped peaks was studied to show the crystal parameters of the CNCs and CNCs based nanomedicines (e.g. crystallinity index, crystallite dimensions and so on). Four of the peaks of (110), (110), (200), and (004) were dealt with Origin Graph software to show the related messages. The diffraction patterns had been adjusted before used. Segal et al had pinpointed the method to calculate the comparative crystallinity of CNCs39. And the equation was as follows:
Where Cr.I. represented the crystallinity index, I200 represented the maximum value of (200) cellulose I reflection, and Iam mean the intensity value at 2θ ≈ 18º. 9
The DLC and DLE of FA/[email protected]@CNCs hybrids nanomedicines were confirmed through the uv-vis absorption spectra with a UV-1800 spectrometer. UV-vis spectrometer was tested using dilute suspensions of CNCs and FA/[email protected]@CNCs hybrids, and the content of DOX was calculated according to standard curves as we did in our previous work 26. Dilute solution was scanned at 480 nm (wavelength). Notably, the interference absorption of CNCs at wavelength 480 nm were deducted before the finally calculation of DOX content of each sample.
The DLC and DLE were calculated according to the following formulas:
The DOX release profiles from FA/[email protected]@CNCs hybrids was studied at 37 ºC in three different media, i.e. (a) acetate buffer, pH 5.5; (b) acetate buffer, pH 6.5; and (c) phosphate buffer, pH 7.4. The concentration of the release medium was 10 mM. FA/[email protected]@CNCs hybrids were divided into three groups (each 1 mL), and speedily devolved to dialysis tubes (molecular-weight cutoff = 1000). The dialysis tubes were dipped into 50 mL homologous buffer solution, which were stirred at 37 ºC. At pre-set time point, 200 μL of release media were taken out for FS, then an equal volume of fresh media was added. The fluorescence intensity was measured. 10
MCF-7 cells cells were purchased from Chinese Academy of Sciences, which were cultured in minimum essential medium (MEM). All of the media were supplemented with 10 % fetal bovine serum (FBS, HyClone, Logan, UT), streptomycin (100 µg/mL) and penicillin (100 g/mL). All cells were incubated at 37 ºC in a humidified 5 % CO2 atmosphere. The confluent cells were dissociated using a pre-warmed trypsin solution at 37 ºC.
2.7 Cell proliferation inhibition
The cell proliferation inhibition of DOX·HCl and FA/[email protected]@CNCs was estimated by MTT assay against MCF-7 cells that were pre-incubated (8 × 103 cells / well) in 96-well plates. The cells with 180 μL of culture medium per well were laid in constant temperature incubator (Thermo, USA). Overnight, the same amount of fresh medium substituted the old one. Subsequently, 20 μL culture media containing of DOXHCl and FA/[email protected]@CNCs hybrids with different concentrations (final equivalent DOXHCl concentration 0.034, 0.067, 0.135, 0.270, 0.539, 1.079, 2.157, 4.314, 8.628 and 17.256 mg·L-1) were added. Thereafter, cells were incubated with DOX·HCl and FA/[email protected]@CNCs nanomedicines for another 48 h. New culture media containing MTT solution (10 μL/well 5 mg·mL-1) was added. 4 h later, 50 μL/well of pyrolysis solution was added to the 96well plates. An automated BIO–TEK microplate reader (Powerwave XS, USA) was used to test the absorbance (wavelength = 570 nm). Wells treated with 200 μL of PBS were used as a blank (ODblank), and cells only treated with 200 μL of culture medium were used as a control (ODcontrol). The cell viability was calculated as follows: 11
2.8 Fluorescence Microscope Images (FMI) and Flow cytometry (FCM)
MCF-7 cells were pre-incubated in a 6-well plate (2×105 cells/well) with 2 mL of complete culture medium per well in constant temperature incubator (Thermo, USA), separately. After incubation for 24 h, the culture media were replaced with new culture media containing DOXHCl or FA/[email protected]@CNCs hybrids of the same DOXHCl concentration (5 mg·L-1). Forty minutes later, the culture media were withdrawing, which were used for the FMI. Cells were washed with pH 7.4 PBS and stained with Hoechst 33342 (10 mg L-1). The FMI were captured on inverted fluorescence microscope (Olympus, TH4-200 with Olympus U-HGLGPS). The other half of the 6-well plates were dealt for the FCM, which were dealt with digestion, centrifugation and washing to dump the nanomedicines or DOX. The cells were collected and analyzed by FCM (Guava easy Cyte 6HT2L, USA). During the experiments, baseline was obtained from the blank control group (cells cultured with normal medium). Experiments mentioned above were performed for three times to reduce errors.
Results and discussion
Nanomedicines present the state-of-the-art laboratory, scientific and clinic aspects of nanotechnologies, nanomaterials, and tools for medical applications. Developing high-performance nanomedicines with optimized physicochemical properties (i.e. active targeting ability and robust preparation method) are critical to find new treatments with various vicious diseases, especially, the
The successful reparation of CAD was descripted and shown in supporting information Figure S2. FA/[email protected]@CNCs hybrids had been developed via the LbL assembly, whose molecular-scale stratified structure was showed in Scheme 2 and in supporting information Figure S1. CNCs with negative charge were the anchors of the hybrids, PEI with positive charge was the intermediary layer and negatively charged CAD and FA were absorbed at the outermost layer of FA/[email protected]@CNCs hybrids. Notably, large amount of PEI was absorbed onto the surface of CNCs, resulting in charge
reversal of the CNCs hybrids. Then, chemotherapeutic DOX was loaded to the pH-responsive hybrids in the form of CAD compound.
Scheme 3: Schematic procedure for FA/[email protected]@CNCs hybrids nanomedicines via LbL assembly: dispersion of the charged anchor in correspondent ionic solution, alternating with centrifugation and washing steps.
The electrostatic interaction based LbL assembly method was employed to construct the FA/[email protected]@CNCs hybrids. The constructional routs were illustrated in details in Scheme 3. As shown in scheme 3, FA/[email protected]@CNCs hybrids were constructed through the most exploited buildup mechanism of LbL adsorption driven by electrostatic interaction of oppositely charged building blocks (i.e. CNCs, PEI, CAD and FA). First, CNCs with negative charge were immerged into polycation PEI solution to form [email protected] hybrids. PEI molecules saturated the negatively charged CNCs resulting in charged-reversal from negative charge to positive charge. There is no aggregate formed after putting PEI and CNCs together. Dynamic light scattering (DLS), atomic force microscopy 14
(AFM) and optical photos were used to confirm the phenomenon. As shown in Figure S14, both DLS and AFM observations confirmed good dispersing of [email protected] nanoparticles. In Figure S15, both the dispersion systems of CNCs and [email protected] showed off-white color fluidic state without any aggregates. Figure S16 is optical photo for the preparation of FA/[email protected]@CNCs hybrids nanomedicines, which confirmed that there were no any aggregations during the whole procedure again. Second, after several times of centrifuge and washing, the deposition (i.e. [email protected] hybrids) was dispersed into the CAD solution. With negative charge, CAD molecules were absorbed onto the surface of the [email protected] hybrids through electrostatic assembly. Then, FA was absorbed onto the surface of the [email protected]@CNCs hybrids as CAD did. Finally, after several times of centrifuge and washing, the targeting hybrids FA/[email protected][email protected] were collected from the deposition. In addition, their photos under normal conditions as displayed in Figure S3 and S16 had verified the successful preparation of FA/[email protected]@CNCs. As shown in Figure S16, the orange-red precipitate was just the target hybrids nanomedicines.
CNCs, and FA/[email protected]@CNCs were measured and depicted in table 1. According to table 1, under the normal condition (pH 7.4), the surface charge studies indicated that the average -potential of CNCs was negative (i.e. -22.6 ± 2.0 mV). In contrast, the absolute average zeta potential value of FA/[email protected]@CNCs hybrids was positive (i.e. 28.0 ± 2.5 mV). The strongly negative zeta potential of CNCs decreased with the addition of PEI. The strongly positive surface charge of these nanoparticles confirmed that the CNCs were capped by large amount of PEI. These results were consistent with the Scheme 3, and the -potential reversion also confirmed the successful preparation of FA/[email protected]@CNCs hybrids via LbL assembly. According to table 1, comparing to the normal condition (pH 7.4), the average -potentials of CNCs and FA/[email protected]@CNCs hybrids stayed relatively stable at the acid condition (pH 6.5). These results should be caused by the proton receptivity of sulfate groups on the CNCs and PEI, which confirmed the successful preparation of FA/[email protected]@CNCs hybrids again. Moreover, it had been determined that the proton sponge polyplex PEI possessed high density of amino groups in the branched scaffold, which were protonable in lysosome (pH 4.5–5.5). The pH buffering behavior named as ‘‘proton sponge” effects would promote the cellular uptake and endosomal escape of nanomedicines. Their pH buffering behavior had been determined by non-aqueous titration (supplementary Figure S12), and their influence on the cellular uptake had been studied in Figure S13 in the supporting information.
Figure 1. (a) FT-IR spectra of (A) CNCs, (B) FA, (C) DOX, (D) CAD, (E) PEI, and (F) FA/[email protected]@CNCs. (b) UV-vis absorption spectra of (A) CNCs, (B) FA, (C) DOX·HCl, (D) FA/[email protected]@CNCs, and (E) CAD.
In addition, FA/[email protected]@CNCs hybrids nanomedicines were also determined by the FT-IR and UV (Figure 1). As Figure 1a exhibited, (E) PEI produced the peaks at 3362 cm-1, 1565 cm-1, 1482 cm-1, and 815 cm-1. The medium intensity absorption peaks that arose near 3365 and 3200 cm-1 belonged to primary amine N-H of PEI. The FT-IR spectra of FA/[email protected]@CNCs (F) did not differ obviously, due to the absorption bands of the additional -NH2, and -CH2- groups of PEI (E) were covered by the broad -OH and 18
-CH stretching bands of cellulose at 3200 ~ 3600 and 2900 cm-1, respectively. The presence of signals at 1425, 1160, 1113, and 895 cm-1 indicated that the CNCs (A) kept their pristine nanocrystal form. The characteristic absorptions of amide bands of CAD (D) were amide I band (1635 ~ 1618 cm-1), amide II band (1578 cm-1) and amide III band (1279 cm-1). FA (B) showed the characteristic absorptions peak at 1656 cm−1. Similarly, the intensity of the characteristic adsorption at 1650 cm−1 in FA/[email protected]@CNCs increased, due to the introduction of FA. The presence of those bands in the FA/[email protected]@CNCs spectrum confirmed that the CNCs based high-performance nanomedicines were successfully prepared. These results were also confirmed by UV-vis spectrophotometer. As displayed in Figure 1b, the characteristic absorption peaks of DOX and FA were at 480 nm and 365 nm 40, respectively. The spectrum of FA/[email protected]@CNCs (F) showed a similar characteristic absorption peaks as DOX did. However, CNCs (A) show no distinct absorption peaks among 200 - 600 nm. Altogether, the robust FA/[email protected]@CNCs hybrids were simply constructed by the easy, efficient extremely versatile way of LbL assembly. According to their characteristic UV absorption peaks at 480 nm (DOX) and 365 nm (FA), standard curves had been made and exhibited in supporting information Figure S17 and S18, respectively, and their molar ratio was 2.40. 3.2 Physicochemical properties of the CNCs based hybrids
The DLC and DLE of the CNCs based hybrids
Table 2. Characteristic drug delivery features of CNC-based hybrids
a: DLC was the abbreviation of drug loading capacity and DLE was the abbreviation of drug loading efficiency.
DLC and DLE of the FA/[email protected]@CNCs hybrids were determined by the FS method according to the formulations as shown in Equation S4 and Figure S5. The DLC was calculated according to a standard curve, and the average amount of DOX absorbed onto FA/[email protected]@CNCs hybrid was 11.3 wt% (DLC) and 19.4 % (DLE). Traditionally, the modification of CNCs utilized chemical conjugation onto their surface through heterogeneous reactions. The external heterogeneous reactions on CNCs surface were not efficient, which directly restricted DLC of the CNC-based nanomedicines. It had been report that the CNCs had high surface area ranging from 50 to 200 m2/g 2 9 41. Taking advantage of their high surface area, the DLC of CNC-based hybrids had been significantly