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Overcoming multidrug resistance through the synergistic effects of hierarchical pH-sensitive, ROS-generating Nanoreactors Ranjith Kumar Kankala, Chen-Guang Liu, Ai-Zheng Chen, Shi-Bin Wang, Peiyao Xu, Lokesh Kumar Mende, Chen-Lun Liu, Chia-Hung Lee, and Yu-Fang Hu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00569 • Publication Date (Web): 07 Sep 2017 Downloaded from http://pubs.acs.org on September 13, 2017

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ACS Biomaterials Science & Engineering

1

Overcoming

multidrug

resistance

through

the

2

synergistic effects of hierarchical pH-sensitive, ROS-

3

generating Nanoreactors

4

Ranjith Kumar Kankala†,‡,*, Chen-Guang Liu†, Ai-Zheng Chen†,‡, Shi-Bin Wang†,‡, Pei-Yao Xu†,

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Lokesh Kumar Mende±, Chen-Lun Liu±, Chia-Hung Lee±, and Yu-Fang Hu#

6

†

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China

8

‡

Fujian Provincial Key Laboratory of Biochemical Technology, Xiamen 361021, P. R. China

9

±

Department of Life Science and Institute of Biotechnology, National Dong Hwa University,

Institute of Biomaterials and Tissue Engineering, Huaqiao University, Xiamen 361021, P. R.

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Hualien- 97401, Taiwan

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#

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Taiwan

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*Corresponding Author:

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Ranjith Kumar Kankala

15

E-mail: [email protected] Tel./fax: +86 592 616 2326

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ORCID: 0000-0003-4081-9179

Pharmaceutical Drug Delivery Division, TTY Biopharm. Company Limited, Taipei 11469,

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Abstract

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Recently, multidrug resistance (MDR) has become a major clinical chemotherapeutic burden

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that robustly diminishes the intracellular drug levels through various mechanisms. To overcome

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the doxorubicin (Dox) resistance in tumor cells, we designed a hierarchical nanohybrid system

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possessing copper-substituted mesoporous silica nanoparticles (Cu-MSNs). Further, Dox was

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conjugated to copper metal in the Cu-MSNs framework through a pH-sensitive coordination link,

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which is acutely sensitive to the tumor acidic environment (pH-5.0-6.0). In the end, the nanocarrier

24

was coated with D-α-Tocopherol polyethylene glycol 1000 succinate (TPGS), a P-gp inhibitor-

25

entrenched compact liposome net for obstructing the drug efflux pump. Copper ions in the

26

framework synergize the anti-tumor activity of Dox by enhancing the intracellular reactive oxygen

27

species (ROS) levels through a Fenton-like reaction-mediated conversion of hydrogen peroxide.

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Furthermore, intracellularly-generated ROS triggered the apoptosis by reducing the cellular as well

29

as mitochondrial membrane integrity in MDR cells, which was confirmed by the mitochondrial

30

membrane potential (MMP) measurement. The advancement of the design and critical

31

improvement of cytotoxic properties through free radical attack demonstrate that the proposed

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hierarchical design can devastate the MDR for efficient cancer treatment.

33 34

Keywords: multidrug resistance, copper, doxorubicin, synergism, liposome, mesoporous silica

35

nanoparticles


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1. Introduction Cancer is one of the leading causes of death, accounting for millions of deaths annually due to their uncontrolled proliferation rate, which has been upsetting the healthcare professionals globally.1 More often, the conventional chemotherapy using various drugs and their combinations has been commonly practiced after surgery and concomitant radiation therapy.1-2 However, this traditional therapy is not quite efficient due to multidrug resistance (MDR) attained by cells, either by cell surface efflux pumps or by anti-apoptotic protein synthesis for self-defense and others.3-6 Numerous cases of cancer exhibit the MDR phenotype, which restrict the entry of drug molecules through various mechanisms.7 Undeniably, MDR significantly affects the therapeutic dosage regimen, and in return high doses of drugs or multiple drugs for combination therapy are utilized, which results in adverse effects clinically.8 These MDR-associated problems have been motivated the researchers to develop the innovative formulations such as utilizing targeting ligands, MDR inhibitors as drugs for controlled drug release approach, and others,7, 9-13 however, their clinical applicability is limited. Doxorubicin (Dox) is among others used solely or in combination with various conventional therapy regimens for a broad range of cancers.12 This broad-spectrum anti-tumor drug has been commonly utilized in the breast cancer treatment, nevertheless, short in vivo half-life, lack of specificity and selectivity of tumor cells and poor solubility, substantially limited its clinical application.14 Also, Dox is not distinctive to MDR,15 however, few researchers have been endeavored to inhibit the MDR of Dox using multi-functional carriers7,

9-12, 15-22

such as

polymers,23-25 and others,16, 26-30 but still, it remains bizarre. Remarkably, nanotechnology has evidenced the rapidly growing interest of researchers in solving the issues-associated with conventional therapy in medicine. Nanoparticle-based 3 ACS Paragon Plus Environment


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approaches are more promising to significantly improve the therapeutic outcome of drugs for oncological diseases by offering many attractive features such as improved aqueous solubility, providing safeguard in the harsh environments, targeted-delivery, and pH-triggered release, among others.1-2, 16, 31-33 Among all inorganic-based nanoparticles, mesoporous silica nanocarriers (MSNs) have garnered the significant attention of researchers ever since their invention by Mobil group scientists34-35 due to their well-defined structure with high surface area, tunable pore size and pore volume for efficient and desirable drug cargo loading, surface functionalization, biocompatibility, exceptional stability, and biodegradability.35-40 These well-known excellent properties of MSNs drove them to utilize not only for controlled drug release but also for delivering various other biological moieties such as a peptide, amino acid, DNA, siRNA.35, 38 Preceding reports on MSNs enunciated that the drug loading capacity typically depends on the affinity between drug and framework, and is managed through weak interactions in a silica pore.36, 41 However, the loading amount is very low, concerning physical adsorption in the pores and their fast releasing ability. To overcome this limitation, we impregnated the copper metal in the mesoporous framework, for enhancing the drug loading amount through coordination interactions, which also facilitates the drug release precisely in the acidic environment of the endosomes. Previously, few attempts have been moderately successful in surpassing MDR using various nanocarriers.

9-10, 12, 15-17, 20, 42-47

In this paper, we designed an innovative hierarchical carrier

utilizing MSNs co-impregnated with the copper metal in the silica framework (Cu-MSNs), which facilitates coordination interactions to immobilize Dox for its pH-sensitive release (Figure 1), in the acidic endosomal environment. In addition, liposome net on the Cu-MSN surface facilitates the incorporation of P-gp inhibitor, TPGS, which is efficient in obstructing the MDR. Subsequently, we investigated the in vitro pH-sensitive Dox release study and anticancer activity


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of Dox in both Dox-resistant and -sensitive cell lines. Eventually, the antiproliferative effects were confirmed through apoptosis by measuring mitochondrial membrane potential (MMP) in MDR cell line.

Figure 1. Schematic illustration of synthesis and cell internalization of designed hierarchical nanoformulation elucidating the delivery of Dox and plausible mechanism of surpassing MDR.



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2. Experimental Details 2.1. Reagents and chemicals. All the reagents, solvents and chemicals were purchased commercially. Doxorubicin hydrochloride (Dox), Ammonium hydroxide (NH4OH) (30%), Cetyltrimethylammonium bromide (CTAB), Copper nitrate trihydrate (Cu(NO3)2 3H2O), 2’,7’-dichlorodihydrofluorescein diacetate (H2-DCFDA),

4’,6-Diamidino-2-phenylindole

dihydrochloride

(DAPI),

D-α-Tocopherol

polyethylene glycol 1000 succinate (TPGS), Formaldehyde (HCHO), Potassium bromide (KBr) (FT-IR grade), Potassium phosphate dibasic (K2HPO4), Potassium phosphate monobasic (KH2PO4), Sodium phosphate dibasic (Na2HPO4), Sulforhodamine-B (SRB), Sodium hydroxide, and Tetraethyl orthosilicate (TEOS) (98%) were obtained from Sigma. Co. Ltd (St. Louis, MO, USA). Ethylenediaminetetraacetic acid (EDTA), Cholesterol, Trichloroacetic acid and Lecithin (phosphatidylcholine) were purchased from Alfa Aesar (A Johnson Matthey company, Heysham, England). 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(polyethylene glycol)2000 (MPEG-2000-DSPE) was obtained from Lipoid & Co. (Ludwigshafen, Germany). McCoy's5A medium and Fetal bovine serum (FBS) were obtained from GIBCO/BRL Life Technologies (Grand Island, NY, USA). 5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC1 dye) was purchased from Invitrogen Ltd. (Eugene, Oregon, USA). 2.2. Physical characterization. The thermal stability of the carrier and its composition determination were examined using thermogravimetric analysis-differential thermal analysis (TGA-DTA) by TGA Q50 V20, 13 Build 39 (Universal V4.5A TA Instruments). Samples were scanned at ambient to 800 ºC at a rate of 20 ºC/min under the dry nitrogen purge at a flow rate of 20 mL/min. FT-IR spectra were recorded on 6 ACS Paragon Plus Environment

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a Bruker Alpha spectrometer at a specified wavenumber range (4000-500 cm-1), the sample was prepared using a KBr pellet method. The particle size distribution (PSD, nm) and ζ-potential distribution (mV) were measured by dynamic light scattering (DLS) phenomena using MalvernZetasizer Nano ZS 90. The samples were diluted and measured at a concentration of 2 mg/mL in water. The zeta potential distribution of all the samples was recorded after adjusting the pH (~ 6.0) and was obtained by averaging the measurements of three samples in triplicate. Powder X-ray diffraction (PXRD) analysis of the copper-impregnated and naked MSN samples was carried-out by using XPert Pro MPD diffractometer, PANalytical Co. Ltd., Holland, at a low diffraction angle (2-theta from 1° to 6°) using CuKα radiation as an X-ray source (2.2 kW). TEM images were captured on a Hitachi H-7100 instrument operating at 100 kV for surface morphology determination. The structural parameters such as BET final surface area, pore size, and pore volume of nanocarriers were determined by N2 adsorption-desorption isotherms recorded on a Micrometric ASAP-2020 apparatus at 77 K. Initially; the sample was degassed at 10-3 Torr, 80 ºC for 3 h before the adsorption. ESR spectra were recorded using a Bruker-EMX spectrometer equipped with a Bruker TE102 cavity. The microwave frequency was regulated with a HewlettPackard (HP)-5246L electronic counter (2 mW power and 4 G at 100 kHz modulation amplitude). Sample preparation was carried out by placing approximately 25 mg of the sample in a 4 mm quartz tube and analyzed at 77 K. UV-Vis absorbance was recorded on a Genequant-1300 series spectrophotometer. Fluorescence intensity was read using Perkin Elmer's EnSpire Multi-label Plate Reader (Santa Clara California, USA). Fluorescence spectral analysis was performed using a Hitachi-F2700 fluorescence spectrophotometer equipped with a xenon lamp.



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2.3. Synthesis of Cu-MSNs. Well-ordered, uniform-sized Cu-MSNs were prepared using TEOS as the silica source, copper salt (Cu(NO3)2. 3H2O), and surfactant template (CTAB) in the basic medium, following the reported co-condensation method.41 Initially, CTAB (0.58 g) was dissolved in 300 mL of NH4OH (0.51 M) and stirred at 40 ºC. After surfactant dissolution, dilute TEOS (0.2 M in 5 mL ethanol) was added and stirred vigorously for 4 h. Later, copper salt (Si/Cu - 30, 5 mL of ethanol) and concentrated TEOS (1.0 M in 5.0 mL of ethanol) was added and stirred for another 2 h. After 16 h of aging at 40 ºC, nanoparticles were collected by centrifugation (12,000 rpm for 17 min). Subsequently, the surfactant template was extracted by the chemical approach using ammonium nitrate (NH4NO3, 0.3 g) in isopropanol (50 mL), stirred overnight at 85 ºC. Eventually, the products were collected, washed and suspended in EtOH, and denoted as Cu-MSNs. 2.4. Dox immobilization. Copper ions-impregnated in the MSN framework serve as an anchor and result in the formation of coordinated interactions with drugs to increase the drug loading efficiency. Dox molecule is one such a drug possessing a free –NH2 group and the coordination between them is achieved at nearly neutral pH (~6.0). As Dox is insoluble in water, we used methanol as a solvent for effective immobilization of drug and the immobilization procedure is performed as follows.40 Initially, 5 mg of Dox was dissolved in methanol (pH adjusted to ~6.0), then the nanoparticles (100 mg) were suspended in the prepared drug solution and stirred overnight at room temperature. The resultant mixture was centrifuged eventually to collect the nanoparticles and resuspended in ethanol. The sample was denoted as Cu-MSN-Dox. Subsequently, the calculated loading


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efficiency was approximately 10% w/w of Cu-MSNs (absorbance recorded using UV-Vis at λmax -480 nm). 2.5. Liposome-coated Cu-MSN sample. TPGS-entrenched liposome network around the Cu-MSNs was coated by following the reported method with slight modifications.48 Initially, the required amounts of lipid phase with four components lecithin, cholesterol, TPGS and MPEG-2000-DSPE in a molar ratio 7: 2: 2: 1, were dissolved in chloroform (30 mL) and the solvent was evaporated under vacuum to generate a thin lipid film. The film was thoroughly dried under vacuum overnight and eventually hydrated by adding Cu-MSN-Dox nanoparticles (75 mg) dispersed in 25 mL of water. The mixture was thoroughly agitated for an hour, and subsequently, bath sonicated for 30 min at 45 °C to generate small unilamellar vesicle (SUV)-coated around Cu-MSNs. This sample is denoted shortly as LipoCu-MSN-Dox. 2.6. In Vitro drug release study. Dox release from Lipo-Cu-MSN-Dox was determined at various pH values mimicking the physiological fluids viz., phosphate-buffered saline (PBS) at pH-5.0 (tumor environment) and 7.4, (physiological fluid such as blood). 5 mg of dried sample was suspended in the respective buffers separately to simulate the release behavior, placed on a rotary shaker (37 ºC, 150 rpm). Aliquots of the sample from the respective buffers were collected at regular intervals and analyzed by UVVis spectrophotometer at λmax-480 nm. The release study was continued by replenishing the respective fresh simulated fluids at their corresponding time points. The cumulative release percentage of the drug was determined periodically.



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2.7. Cytotoxicity studies. 2.7.1. Cell culture and reagents The in vitro anticancer studies were performed using human uterine sarcoma, Dox-resistant tumor (MES-SA/Dx-5) and the colon carcinoma (HT-29) cell line. The cell culturing processes are diverse, where the HT-29 cell line was cultured in RPMI-1640 medium, and the MDR (MESSA/Dx-5) cell line was maintained in McCoy’s-5A medium containing 1.5 mmol/L of L-glutamine. Both the media were supplemented with 10% (v/v) fetal bovine serum and antibiotics such as penicillin (100 units/mL) and streptomycin (100 μg/mL). The culturing process was performed in a humidified incubator maintained at 37 °C in 5% CO2. 2.7.2. Sulforhodamine B (SRB) assay. The in vitro cytotoxicity of designed nanocarriers was determined using an SRB assay by following the reported method.49 Cells were seeded into a 96-well plate at a density of 1x104 cells/well and incubated at 37 °C for 24 hours. At first, the treatment plate is divided into three groups, i.e., two control groups and one sample treatment group. Cells in one of the two control groups were fixed in situ using trichloroacetic acid (TCA, 25 µL, 50% (w/v) in water) to determine the cell number (T0) in order to synchronize the cell number of treatment groups as well as control group (CTL), which received medium alone. Further, the cells in the treatment groups (Tx) were subjected to different concentrations of nanoparticle samples suspended in the serum-free medium. Post-incubation for 4 hours, the same volume of media supplemented with 20% FBS was added to all groups of cells except T0 for nourishment and incubated for 20 hours. Further, the cold TCA (50 µL) was added to the remaining groups (Tx groups and the control group) for cell fixation and incubated at 4 °C for an hour. Later, the medium in all the wells was pirated, washed thrice with 10 ACS Paragon Plus Environment

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dd-H2O and then air-dried for 12 hours. Then, the prepared SRB solution (100 µL of 0.4% (w/v) in 1% acetic acid) was added to each well and incubated. Further, the unbound SRB was removed, and the bound dye was subsequently solubilized by the addition of 10 mM of the trizma base (pH10.5). The absorbance was measured eventually at 515 nm, and the percentage of growth inhibition was calculated using the formula: 100−[(Tx −T0)/(CTL−T0)]×100 (when Tx≥T0). 2.7.3. Reactive oxygen species (ROS) assessment. Intracellular ROS levels play a significant role in cell assassination. These levels were monitored using the free radical sensor/probe, H2-DCFDA. The deacetylated fluorescent product, i.e., dichlorofluorescein (DCF) of this hydrophobic cellular permeable probe was generated by the catalysis of cellular esterase in the presence of free radicals. The ROS levels were determined using the following method.50 Cells were seeded at a density of 1x104 cells/well into 96-well culture plates and incubated overnight for proper cell attachment. Later, various concentrations of nanoparticle sample suspended in the FBS-free medium were treated and incubated for 4 hours alongside with the controls, i.e., media alone (negative control), H2O2 (positive control) and pure Dox. Further, the dye (H2-DCFDA, 20 μM) was added to wells and incubated for 30 min at 37 °C. Eventually, cells were given a wash with PBS, and DCF fluorescence was analyzed using Elisa reader. The assay was performed in triplicate for three independent experiments. 2.7.4. Cellular uptake study The drug delivery efficacy of metal-impregnated nanoparticles and its successive liposomecoated conjugates was investigated using cell uptake study through the visualization of nanoparticles in confocal laser scanning microscopy (CLSM).48 The anti-tumor drug; Dox has an added advantage of possessing auto-fluorescence property. Herein, Dox-resistant cells (MES11 ACS Paragon Plus Environment


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SA/Dx-5), after 80 % of confluence were harvested and seeded into the CLSM-specific dishes at a density of 1 x 105 cells per well and incubated for 24 h for proper cell attachment. Liposomecoated and uncoated Cu-MSN-Dox nanoconstructs suspended in FBS-free media were incubated with the cells for 4 hours, and then the cells were fixed with 3.7% of paraformaldehyde and incubated for 10 minutes with 0.5% of Triton X-100 for cell wall dissociation. Further, the nucleus was stained with DAPI (0.1 mg/mL), all the additions were intermittently washed thrice with PBS. Eventually, the images of stained cells were captured using CLSM. 2.7.5. Morphological analysis of mitochondria. An apoptosis indicator, MMP was measured ratiometrically using the dual emission potential sensitive dye, JC-1 stain (5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl benzimidazolyl carbocyanine iodide) through color variation observed in the cells after treatment.51-52 This indicator signifies both qualitative as well as quantitative measurements of MMP and organelle integrity. Cells were seeded at a density of 1x104 cells/well and incubated overnight for proper cell attachment. Later, the cells were treated with designed nanocarriers, concomitantly MMP levels were compared with the control and pure Dox. After 4 hours, 10% FBS in the medium was added and incubated for further 20 hours. Consequently, the cells were incubated after adding JC-1 dye (5 μM) for 30 minutes; the morphological analysis was performed by observing under the fluorescent microscope representing the variation in dye aggregates, which demonstrates the deviations in MMP. In addition, the J-aggregates were quantified by measuring the fluorescence at corresponding wavelengths (530 nm (green) and 630 nm (red)) in Elisa reader.


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3. Results and Discussion The material design of our multi-functional innovative nano-formulation is depicted in Figure 1. The chief aim of the design is to overcome Dox resistance and assassinate MDR cells through a synergistic effect. Initially, we have synthesized well-ordered, hexagonal, metal-impregnated MSNs (Cu-MSNs) by co-condensation of TEOS and copper salt in a basic medium using a CTAB template for mesopore generation. The co-impregnated metal viz., Cu(II) in the mesopore framework anchors the drug through a coordination linkage, which also serves as a pH-sensitive trigger to deliver the drug in the low pH environment of the endosomes.41 Metal impregnation in the framework also facilitated the high drug loading efficiency. Further, the Cu-MSNs were coated with liposome network using neutral and slightly positive charge bearing lipids to improve the cellular internalization of nanocontainers, facilitating the enormous likeliness of surpassing MDR. Cu-MSNs are arranged/remained in the hydrophilic core of the liposome, due to the surface hydroxyl groups in the mesoporous framework, that favor the interactions with the liposome. The components and their respective concentrations of bilayer formation were optimized such that it yields the spherical small unilamellar vesicles encapsulating the nanocarriers for the internalization of MSNs. In addition, the inherent character of lipids, their thermodynamic phase properties, and self-assembling characteristics influence the hydrophobic sections to form spherical bilayered vesicles.53 At first, the synthesized nanocarriers were physically characterized using various techniques such as Fourier transform infrared spectroscopy (FT-IR) recordings for functional group identification, thermogravimetric analysis (TGA) for weight loss determination, DLS measurements for determining the particle size and surface charge distribution, UV-Vis detection and fluorescence measurements for loading amount calculation and Dox conjugation determination respectively, powder X-ray diffraction studies and electron spin resonance (ESR) 13 ACS Paragon Plus Environment


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measurements for clarifying the copper impregnation, and nitrogen adsorption-desorption measurements for the determination of structural properties and then in vitro Dox release at various pH-values is performed to verify pH-sensitive Dox release through coordination bond dissociation. Further, we demonstrated the anti-cancer studies using both Dox-resistance/-sensitive cell lines for the determination of the IC50 values. Eventually, the ROS levels are measured by using the H2DCFDA,50 and the resultant apoptosis confirmation by the MMP measurement.52 3.1. Physical characterization FT-IR spectra were chronicled to elucidate various organic functional groups immobilized in the silica framework (Figure 2). A strong and broad band at 3450 cm-1 is due to O-H stretch and surface Si-OH as well as absorbed atmospheric water molecules (Figure 2a-d). The surface silanol groups Si-O-Si at 1080, 792 cm-1 and Si-O-H at 966 cm-1 characterizes the formation of stable silica framework. High-intensity peaks of C-H stretch (Figure 2a) at around 2850-2925 cm-1 and C-H deformation peak at 1430 cm-1 represents the surfactant template in pristine Cu-MSNs. After surfactant template removal by the chemical extraction method (ammonium nitrate in isopropanol),41 the characteristic peaks of CTAB were disappeared (Figure 2b) and the characteristic peaks ascribed to silica framework remained same with no changes demonstrating that it is quite stable during the extraction process. Further, Dox immobilization in the Cu-MSNs framework resulted in various peaks such as a broad peak centered at 1740 cm-1 attributed to saturated aliphatic carbonyl group, a peak at 1560 cm-1 accredited to amine group, and peaks attributed to C-H vibration at 2930 and 1460 cm-1 (Figure 2d), which were in agreement with the characteristic peaks obtained in pure Dox sample (Figure 2c).54-55 Moreover, we observed a slight shift in the peak ascribed to N-H vibration from 1527 to 1560 cm-1 after dox immobilization, confirming the establishment of coordination interactions. Comparatively, the peak intensities of


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Dox seems very low due to higher intensity peaks of the silica framework. Though the Dox-loaded Cu-MSNs sample resulted in the low-intensity peaks, the drug loading amount was confirmed by thermogravimetric analysis (Figure S1) and UV-Vis determinations. (a)

(Si-O-Si)

1080 792 3450

2852

966

(O-H stretch)

2925

(Si-O-H)

(C-H stretch)

(b)

% Transmittance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(c) (C=O)

1717

2932 (C-H)

1467 (C-H) 1527 (N-H)

(d)

1745

(C=O)

2930 (C-H)

1560 1460

(N-H)

(C-H)

(e) 2855 1741 1381

2922 (C-H)

(C-O-) (PO4) 1225

4000

3000

2000

1000

0

Wavenumbers (cm-1) Figure 2. FT-IR spectra of (a) pristine Cu-MSNs, (b) surfactant template-extracted CuMSNs, (c) pure Dox, (d) Cu-MSN-Dox, and (e) Lipo-Cu-MSN-Dox. Eventually, the Dox-loaded Cu-MSNs were coated with liposome network, which epitomized peaks at 1381 and 1741 cm-1 attributed to –C-H bending and the –C-O stretch of lecithin ester, respectively. In addition, a sharp peak at 1225 cm-1 embodies the PO4 stretch of lecithin and DSPE. A few sharp bands at 2922, 2855 illustrate the C-H stretch of cholesterol and 15 ACS Paragon Plus Environment


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aliphatic hydrocarbon chain of PEG in TPGS (Figure 2e). These characteristic observations elucidate the preparation of Cu-MSNs along with successful functionalization processes of substantial Dox loading and liposome coating around Cu-MSNs. The drug loading, as well as the thermal properties such as stability of Cu-MSNs and its successive nanoconjugates, were robustly corroborated by TGA. Figure S1A illustrates the TGA curves demonstrating the events of weight loss, i.e., stages of degradation of designed nanoconjugates. The elucidated individual thermal behaviors of all the samples were varied significantly. At the outset, the first event of weight loss before 100 °C, is ascribed to the residual water in all the samples. In the as-synthesized Cu-MSNs sample (Figure S1B-a), the second event of weight loss is at around 230 °C, due to the early degradation of organic surfactant template, and the same event vanished in the surfactant template-extracted Cu-MSNs sample, demonstrating the removal of the surfactant template (Figure S1B-b). Further, the Cu-MSNs-Dox sample resulted in a significant weight loss event at 375 °C, (Figure S1B-c), corresponding to the Dox loading amount in nanocarriers (~10%), which is in agreement with the UV-Vis measurements (See Dox immobilization segment under Methods section). The actual degradation temperature of Dox is at around 200-250 °C (data were not shown),12 but the degradation temperature shifted towards the right when immobilized in Cu-MSNs, signifying not only the Dox immobilization in the pores of Cu-MSNs but also represents the enhancement of its stability. Furthermore, the degradation event in the liposome-coated Cu-MSNs sample started early at around 250 °C, and this broad event continued till 600 °C. In addition, we also observed the upshift in the degradation temperature of Dox, which overlapped in an event at 430 °C, demonstrating that the enhanced thermal stability with respect to the strength of interactions between the molecules. Henceforth, all these incidents


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of weight loss successfully confirm that the drug cargo was loaded in the mesopores of Cu-MSNs and the liposome network was coated hierarchically around the Cu-MSNs.

Figure 3. A) BET isotherms of Cu-MSNs and its successive modified samples and their corresponding B) pore size distribution curves. (a) pristine Cu-MSNs, (b) surfactant templateextracted Cu-MSNs, and (c) Cu-MSN-Dox. The structural parameters/textural properties such as final surface area, pore size, and pore volume of Cu-MSNs, as well as its successive conjugates, were elucidated based on the N2 adsorption-desorption isotherms by Brunauer-Emmett-Teller (BET) analysis (Figure 3, Table 1). The samples were degassed applying vacuum (80 ºC for 3 h) before (BET) analysis. All the isotherms displayed type-IV gas adsorption curve, according to IUPAC classification.41 The 17 ACS Paragon Plus Environment


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surfactant template-extracted Cu-MSNs sample has shown a drastic increase in its final BET surface area (1283 m2/g) (Figure 3A-b) than the pristine Cu-MSNs (809 m2/g) (Figure 3A-a), signifying that the surfactant template was extracted and resulted in nano-sized pores. In addition to textural properties determination, BET analysis can be correlated to evaluate the particle size. Previous literature has revealed that the final BET surface area is indirectly proportional to the particle size. In a way, the materials with the larger surface area are smaller in size and vice versa.56 Similarly, the final surface area of our sample (Cu-MSNs) is quite higher than the reported value, indicating that the particle size of Cu-MSNs is small. Further, the Dox-loaded sample resulted in a decrease of final BET surface area, demonstrating that the immobilized drug molecules occupied the space in the pores (Figure 3A-c). Pore size distributions (Figure 3B) of the respective samples followed the same pattern as the surface area did, with an increase in pore size and pore volume after extraction (Figure 3B-b) and reduced after loading Dox in Cu-MSNs (Figure 3B-c). The BETanalysis has no chance to determine the exact BET surface area of liposome-coated Cu-MSNs because liposome has a less scope for the adsorption of nitrogen, which eventually may result in the data manipulation.48 DLS measurements (Table 1) gave the hydrodynamic diameter and zeta potential values of Cu-MSNs and its successive conjugates. The average hydrodynamic diameter of pristine CuMSNs, surfactant template-extracted and Dox-loaded Cu-MSNs is around 100-150 nm. Further, the liposome-coating around Cu-MSNs resulted in an increase in its size, at around 300 nm, due to slight aggregation during measurement. From the TEM images, the particle size distribution of all the conjugates was more or less in agreement with the DLS measurements. However, the size of the liposome-coated sample is lesser compared to DLS observations. In addition, we calculated the dispersity indices of all the samples concerning TEM and DLS measurements, and the values


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are in the range of 0.1-0.2, demonstrating that they possess excellent suspendability and are suitable for biomedical applications. It is evident from the TEM images that a very thin layer of liposome coat around Cu-MSNs demonstrated the formation of SUVs and the thickness of the coat is at around 10-20 nm. In general, MSNs possess negative surface charge (-22±0.2 mV at pH-6.0) after surfactant removal,41 but it was less negative (-15±0.45 mV) in the case of Cu-MSNs, due to copper ions in the framework. Copper ions may also support in establishing electrostatic interactions with the negatively charged biological membrane, which is beneficial for synthetic biologists in activity based assays. After immobilizing Dox, the zeta potential value increased towards the positive end and eventually shifted back towards the negative end, after liposome coating, due to cholesterol and TPGS in the composition. However, the charge of liposome does not have any influence on the cellular internalization process of nanocarriers, as the liposome entangles with the cell membrane lipids and deliver the nanocarriers inside the cells through endocytosis.48 Table 1. The structural (BET) and dynamic light scattering (DLS) parameters of Cu-MSNs and their successive conjugates. Sample

BET surface area

Pore volume

Pore size

Particle diametera

(m2/g)

(cm3/g)

(nm)

(nm)

Dispersity

Zeta potentiala (mV)

DLS

TEM

DLS

TEM

Pristine Cu-MSNs

809

1.32

2.1

142±13

125±16

0.09

0.13

32±1.25

Cu-MSNs

1283

2.2

2.4

148±14

139±14

0.10

0.10

-15±0.45

Cu-MSN-Dox

1173

1.50

2.2

162±13

135±15

0.08

0.11

-13±0.11

Lipo-Cu-MSN-Dox

N.D

N.D

N.D

328±25

242±31

0.08

0.12

-37±1.10

N.D- Not determined, Abbreviations: DLS- Dynamic light scattering, TEM- Transmission electron microscopy. aAll values are represented as a mean ± standard deviation.



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Powder X-ray diffraction (PXRD) pattern (Figure 4A) of the Cu-MSN sample exhibited a hexagonal mesoporous structure, which is typical of the MCM-41 type with basal reflection peaks (low-angle, 2θ) at 2.41, 4.1 and 4.7 corresponding to (100), (110), and (200) reflection planes of P6mm symmetry (Figure 4A-b).57 This pattern demonstrates the ordered pore structure, large surface area, and the defined size. The peaks corresponding to the typical basal reflection plane (100) and (110) in Cu-MSNs has resulted in a shift in its peaks towards right compared to MSNs (2θ-2.28, 4.0 respectively) (Figure 4A-a), demonstrating the altered mesoporous framework. Figure 4B illustrates the surface morphology from TEM observation of the Cu-MSNs sample. The particles are uniform with an average size around 100 nm, and these results are in agreement with the DLS measurements (Table 1). Further, the liposome coating over Cu-MSNs (Figure 4C) resulted in a clumpy and misty layer surrounding the nanocarriers, confirming the successful deposition of the liposome. In our previous report, we demonstrated the structural characteristics of metal impregnation in the silica framework (Cu-MSNs) by ESR spectra, which resulted in the anisotropic copper (II) signal at 77 K revealing that copper centered systems were coordinated axially with siloxyl groups.41 The g values suggested that the distorted square pyramidal octahedral coordination of copper within the siliceous framework demonstrating the copper (II) complex (Figure S2).58 The fluorescence spectra explored the absorption characteristics of Dox along with its coordination to the mesoporous framework (Figure 4D). The sample specimens were excited at an absorption maximum of Dox (λex: 440 nm) and resulted in maximum emission at around 580 nm (λem)40 (Figure 4D-a). Furthermore, the liposome-coated Cu-MSN-Dox sample (Figure 4D-b) resulted in a fluorescence shift (i.e., blue shift) due to coordination linkage as compared with pure Dox. Moreover, the pH-sensitivity of coordination linkage between copper and Dox was


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demonstrated by suspending the sample in a buffer solution (pH-5.5) for a span of 24 h, which eventually resulted in devoid of fluorescence peak at the respective emission region of Dox (corresponds to the complete release of the drug), confirming that the coordination link is absolutely sensitive to acidic environment in tumor (Figure 4D-c).

Figure 4. (A) PXRD patterns of a) MSNs and b) Cu-MSNs. TEM images of (B) the characteristic hexagonal structure of Cu-MSN-Dox and (C) liposome-coated Cu-MSN-Dox (Arrow showing the liposome coat over the Cu-MSNs in the magnified view), (D) Fluorescence spectra of (a) pure Dox (red line), Lipo-Cu-MSN-Dox sample (b) before (blue line) and (c) after incubation at pH-5.5 for 24 h (black line). (E) Dox release from liposome-coated Cu-MSNs at various time intervals in simulated fluids (phosphate-buffered saline (PBS) at pH-5.5 and 7.4).



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3.2. In Vitro drug release study The in vitro release behavior (Figure 4E) of dox was investigated by measuring the absorbance using UV-Vis spectrophotometer in the respective buffers (i.e., simulation of tumor environment (pH-5.5) and physiological fluids (pH-7.4)). Interestingly, the characteristic pH-sensitive coordination link was disrupted in the acidic environment, and the release of Dox is significantly high, i.e., 10 times more, compared to the physiological buffer at pH-7.4. This pH-sensitive release behavior favors the conveyance of the drug cargo to the tumor environment and prevents the outflow of the drug in the physiological fluids during delivery. 3.3. Plausible mechanism In this strategy, a few sequential steps such as surpassing MDR, pH-triggered delivery, and eventually cancer cell assassination are involved for efficient delivery of drugs. The plausible mechanism of action lying behind the success of the design is the generation of toxic ROS by the synergistic approach. During drug delivery to the tumor, the liposome around Cu-MSNs successfully entangles with the cell membrane for its internalization and delivers the Doximmobilized Cu-MSNs cargo through endocytosis. In a way, TPGS in the liposome network also enhances the cellular uptake.59 Upon endocytosis, the acidic environment in the endosome ultimately weakens and dismantles the pH-sensitive coordination link and triggers the Dox release by its protonation. More often, the detached Dox molecules in the tumor escape the endosomes and generate hydrogen peroxide involving the oxidative events of anthracycline derivatives, which are comparatively higher than the apparent hydrogen peroxide concentration intracellularly. Initially, the released Dox molecules undergo reductive metabolic activation, converted to its semiquinone derivative and generates superoxide anion through one-electron reduction of oxygen. The resultant superoxide further undergoes dismutation reaction in the presence of superoxide


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dismutase and results in hydrogen peroxide (Eq. 1).60 Further, the Fenton-like redox chemistry of copper results in the conversion of hydrogen peroxide to highly toxic free radicals, i.e., hydroperoxyl radicals (Eq. 2) and then eventually to toxic hydroxyl radicals (Eq. 3). These toxic free radicals reduce the mitochondrial membrane fluidity and stimulate the apoptotic cascadesmediated by cytochrome c release upon MMP alteration. The sequence of reactions involved in the generation of free radicals is listed below. Dox + O2 + 2H+→ Dox + H2O2

(1)

Cu2+-MSNs + H2O2 → Cu+-MSNs + HO2∙ + H+

(2)

Cu+-MSNs + H2O2 → Cu2+-MSNs + OH- + OH∙

(3)

Copper is an essential trace element and is a fundamental part of many important enzymes in various vital biological processes. Though it is bound to enzymes in the body, free form of copper is active in the redox chemistry to catalyze the generation of highly toxic ROS in the presence of hydrogen peroxide intracellularly, which is predominantly higher in cancer cells than normal cells for self-defense mechanisms such as autophagy and others. However, it may interfere with the important cellular events at chronic copper overload.61 In this study, the oxidation state of copper is +2 and remains unchanged in acidic pH. However, the change in oxidation state to +1 can be observed in the presence of electron donors such as hydrogen peroxide to catalyze the production of highly toxic free radicals (Eq. 2, 3). 3.4. Cytotoxicity studies 3.4.1. Antiproliferative effects The distinctive anti-proliferation effects of Cu-MSNs and their successive conjugates were studied using Dox-resistant (MES-SA/Dx-5 (Figure 5)) as well as -sensitive (HT-29 (Figure S3)) 23 ACS Paragon Plus Environment


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cell lines. This comparative methodology prominently emphasizes that the Cu-MSNs could reverse MDR and enhances the ROS production and eventually assassinates the drug-resistant cells.

Figure 5. SRB assay of various concentrations of nano-complexes (a) Cu-MSNs, (b) pure Dox, (c) Cu-MSN-Dox, and (d) Lipo-Cu-MSN-Dox for evaluating the cell growth inhibition in MES-SA/Dx-5 cell line. Cells were fixed after 24 hours, stained with SRB, washed with dd-H2O, and subsequently, solubilized before absorbance was read at 515 nm. IC50 values were determined from the graphs.


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Figure 5 depicts the antiproliferative effects of variously designed nanoconstructs measured using SRB assay, Lipo-Cu-MSN-Dox (1.8 µg-NPs/mL) (Figure 5d) and Cu-MSN-Dox (3.5 µg-NPs/mL) (Figure 5c) samples have shown the lower IC50 value compared to the free Dox (22.7 µM/~13.1 µg-Dox/mL) (Figure 5b), signifying the effective surpassing of MDR in cells. The Dox concentration in NPs at IC-50 values with respect to their loading efficiency (i.e., ~10%) is ~0.18 µg-Dox/mL of Lipo-Cu-MSN-Dox and ~0.35 µg-Dox/mL of Cu-MSN-Dox, which is significantly lower compared to pure Dox concentration. The interesting feature of this formulation is the presence of copper ions in the framework, which synergizes the battle by participating in free radical reactions of ROS generation. Moreover, Cu-MSNs-treated cells also resulted in cell death (IC50-121.6 µg/mL) (Figure 5a). The anti-tumor effect of Lipo-Cu-MSN-Dox is higher than that of all other nanocarriers and pure Dox, because of the synergistic effects of loaded Dox, copper in the framework and P-gp inhibitor TPGS in the liposome network, which might influence the drug action by reversing the MDR. In addition to drug-resistant cells, we have observed the antiproliferative effects of the designed nanocarriers in the Dox-sensitive cells (HT-29 cells) (Figure S3). The IC50 values of the synthesized nanocarriers (Cu-MSNs-118.5, Cu-MSN-Dox-3.5, Lipo-Cu-MSN-Dox-2.0 µg/mL) are similar to the resistant cells. However, the IC50 value of pure Dox is much lower (IC50-13.4 µM/~7.7 µg-Dox/mL) than Dox-resistant cells. The antiproliferative effect of Lipo-Cu-MSN-Dox is greater in drug-resistant cells than the sensitive cells, due to overexpressing of MDR-associated proteins and its subsequent obstruction by the nanocarriers resulted in lower IC50 value.



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Figure 6. Graph showing ROS generation using H2-DCFDA in control (media alone), hydrogen peroxide (H2O2 -1 mM), pure Dox (50 μM), Cu-MSNs, Cu-MSN-Dox and Lipo-Cu-MSNDox in MDR cell line (MES-SA/Dx-5 cell line). (*p

Overcoming Multidrug Resistance through the ... - ACS Publications

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