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Augmented anticancer efficacy by si-RNA complexed drug loaded mesoporous silica nanoparticles in lung cancer therapy Fahima Dilnawaz, and Sanjeeb K Sahoo ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.7b00196 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

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ACS Applied Nano Materials

Augmented anticancer efficacy by si-RNA complexed drug loaded mesoporous silica nanoparticles in lung cancer therapy

Fahima Dilnawaz*, Sanjeeb K Sahoo*

Laboratory of Nanomedicine, Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar 751023, Odisha, India

Corresponding Author

Email Address: [email protected] (Fahima Dilnawaz)

:[email protected] (Sanjeeb K Sahoo) Laboratory of Nanomedcine Institute of Life Sciences, Nalco Square, Chandrasekharpur, Bhubaneswar, Orissa, INDIA Phone- 91-674-2302094 Fax- 91-674-2300728

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Abstract:

Lung cancer remains as the major cause of deaths throughout the world. However, with the cutting-edge therapeutic approaches the patient’s survivility remains poor. Nanoparticle mediated targeted gene therapy has surfaced globally for the therapeutic improvement in cancer patients. Recently, mesoporous silica nanoparticles (MSNs) have attracted much attention for the intracellular delivery of siRNA. Further co-delivery strategy has been proposed to minimize the amount of each drug and to achieve the synergistic effect for cancer therapies. In the present investigation we have explored the efficacy of co-delivery of anticancer drugs along with survivin siRNA in the MSNs for a comparative therapeutic efficacy in lung cancer. Our results revealed such combinational approach has provoked enhanced therapeutic efficacy by synergistic drug activity while restraining the action of survivin protein. Thus, we anticipate this emerging approach may be quite beneficial for lung cancer therapy in future.

Key words: Mesoporous silica nanoparticles, siRNA, combination therapy, carfilzomib, gene therapy

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Introduction

Lung cancer is the second most common cancer in men and women in the world and the epidemic of lung cancer is continuing with increased incidence.1 Two broad classes of lung cancers are categorized: non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC). NSCLC accounts for approximately 85 % of all cases of lung cancer. Despite of major advance treatment options in chemotherapy, radiation therapy and patient management, still the 5-year survival rate is nearly 4 %.1,2 In NSCLC, drugs that are used as first-line treatment is often platinum based which is combined with any one of the other drug such as paclitaxel, docetaxel, pemetrexed, etoposide or vinorelbine.3,4 This gold standard of treatment has prolonged many patients’ lifespan and improved their quality of life. Using combination therapy, codelivery of drugs exhibits high therapeutic effects in a synergistic manner and overcome drug resistance and undesirable side effects towards the normal tissues enabling better survival rate. 5,6

Cancer cells incur multidrug resistance (MDR) which is multifactorial and in turn restrain apoptosis and creates hindrance for effective cancer therapy. Broadly ‘‘pump’’ and ‘‘non-pump’’ forms of resistance usually contribute to chemotherapy resistance.7,8 Pump or transport mediated resistance is due to drug efflux through ABC proteins and cytoplasmic vesicles etc, whereas, the basic mechanism of non-pump resistance is activation of cellular anti-apoptotic defense.9 In critical cellular processes and cell signaling pathways the ubiquitin-proteasome system controls the turn-over of regulatory proteins involved and contributes to the pathological state of many human diseases including cancers and are essentially involved in development of tumor drug 3



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resistance.10,11 The proteasome is a multimeric protease complex that is central to the highlyregulated ubiquitin proteasome protein degradation system, which regulates numerous signaling pathways involved in cell proliferation, cell cycle control, apoptosis and moreover these events are dysregulated in malignant cells.12,13 Recently dramatic interest has been drawn towards proteasome as a target for cancer therapy. These agents are able to induce apoptosis and tumor regression either individually or in combination through synergism effect.14 Proteasome inhibition has been extensively investigated as a selective anti-cancer strategy and is validated in clinical trials 15. Inactivation of proteasome function induces increased apoptosis thereby leading for enhanced antitumor effect. Carfilzomib, is an irreversible selective proteasome inhibitor specific for chymotrypsin-like active site, has been illustrated unprecedented benefit in patients with multiple myeloma. Because of its promising anticancer activities and favorable toxicity profile, it has been explored as a potential therapeutic for lung cancer therapy.16,17 Survivin, member of inhibitor of apoptosis protein (IAP) family is highly expressed in almost all types of human tumors. Elevated expression of survivin in lung cancer is associated with tumor progression, angiogenesis, resistance to drug treatment and radiation leading to poor survival of cancer patients.20-22,23 In cancer treatments, different approaches have been employed to target survivin such as small interfering RNAs, antisense oligonucleotides,18 ribozymes,

19

and triplex

DNA.20 However, the RNA interference (RNAi) therapeutics has made noteworthy progress since the first demonstration of gene knockdown in mammalian cells. 21,22 The RNA interference mechanism has been developed for endogenous post-transcriptional gene silencing inside cells. Utilizing this event many researchers have targeted survivin using viral vector mediated siRNA delivery. However, the therapy is curtailed by the barriers for siRNA to reach their intended targets in the cytoplasm for exerting their gene silencing activity. Recently, nanotechnology4


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mediated non-viral vectors based gene delivery system has attracted much attention for better therapeutic efficacy and offers important prospective for inducing specific potent silencing of genetic targets. 23

There is a surge of interest in using inorganic engineered nanoparticles for different biomedical applications. Mesoporous silica nanoparticles (MSNs) have recently engrossed significant attention in biomedical applications due to their advantageous structural properties, such as huge specific internal surface area and pore volume, tunable pore sizes, colloidal stability, and biocompatibility with high drug-loading capacity and ease for surface functionalization for targeted therapy. These attractive features have made MSNs a promising platform for diverse biomedical applications such as biosensing, 24 biocatalyst, 25 bone repair and scaffold engineering,

26,27

bioimaging for diagnostics

28,29

and drug delivery,

30-32

, theranostic. 33

Additionally MSNs-based stimuli responsive system has been designed for drug release triggered by either external or internal environmental stimuli, such as temperature, 34,35 light, 36 enzymatic activity, 37,38 pH, 39 redox potentials. 40,41 When these MSNs are degraded in cells, their size and dispersibility remained unchanged that in turn reduces the biological toxicity. Moreover, the biodegradation product of MSNs, i.e. ortho-silicic acid is a natural compound with low toxicity found in numerous human tissues hence potentiating its biomedical application.

9,42

MSNs are viewed as promising candidates for siRNA delivery as the large surface area provides space and binding sites to accommodate siRNA. For this, MSNs are typically modified with positively charged polymers such as poly-l-lysine (PLL) or polyethyleneimine (PEI) for binding of negatively charged nucleic acids via electrostatic interactions.

43,44

Research interest

continues towards developing molecularly targeted therapeutic strategies for lung cancer. 5



Combined therapy using different approaches has made significant progress in the field of cancer therapy. In a recent study, Zhang et al, has used MSNs as drug delivery vector and loaded it with photosensitizer chlorine e6(C6) and conjugated cisplatin prodrug for synergistic activity to conquer cisplatin resistance against lung cancer.45 In another approach, Lu et al, fabricated a camptothecin loaded redox responsive nanohybrids using gold nanoparticles and MSNs for potential growth inhibitory effect due to higher oxidative stress in lung cancer cells.46 Combination of various therapeutic mode has emerged as a promising strategy for lung cancer treatment. However, the main hindrance for lung cancer therapy is activation of nonpump resistance due to elevation of cellular antiapoptotic defence system like survivin protein. Elevated expression of survivin makes it an appealing target for novel therapies in lung cancer.47 Further, the ubiquitin-proteasome system (UPS) plays a significant role in cell proliferation and survival and the cancer cells take the advantage of the UPS to achieve aberrant growth and exerts resistance to apoptosis. Selective anticancer strategy using proteasome as target has not been explored much. Keeping all the above factors in mind, in the present investigation, we have attempted to co-deliver carfilzomib (a proteasome inhibitor) with anticancer drugs (etoposide or docetaxel) in MSNs targeted with survivin siRNA for a comparative therapeutic efficacy study for lung cancer therapy.

Results and Discussion Physicochemical and material characterization of synthesized MSNs Advances in nanotechnology towards drug delivery have opened up exceptional opportunities for promising drug carrier MSNs for cancer treatment. In this regard, MSNs were synthesized by cetrimonium bromide (CTAB) template method using ammonia and silica 6


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precursor tetraethyl orthosilicate (TEOS). The hydrodynamic diameter of the void-MSNs determined by dynamic light scattering (DLS) indicated average hydrodynamic diameter of ~ 172 nm, with zeta potential of ~ -21 mV. The negative zeta potential of void-MSNs is contributed due to the large amount of negatively charged silanol group present on the surface of MSNs. To the void-MSNs, the anticancer drugs were loaded into the pores either individually or in combinations and their hydrodynamic diameter and zeta potential were slightly increased (Table-S1). Several groups have synthesized MSNs, via different techniques and have witnessed monodispersity with negative zeta potential.

9,48,49

An efficient nanocarrier should possess a

greater drug loading capacity so that there will be reduction of dose in administration. In this regards, to investigate the efficacy of various single drug formulations of docetaxel (DOC), etoposide (ETO) and carfilzomib (CAR) loaded MSNs and combined drug formulations of (DOC + CAR), (ETO + CAR) in MSNs were prepared. The entrapment efficiency of different drug either in single formulation or in combined formulation was estimated by HPLC and the results indicated that each drug was efficiently entrapped in single drug formulations as well as in combined drug formulations (Table-S1). The slight difference in entrapment efficiency of single drug in MSNs may be attributed to the chemical structure of the drug and interaction with the SiO2. The stronger the interactions, the better the entrapment efficiency. It is a well-documented fact that targeted delivery and controlled release features are the major contributing factor for providing better cytotoxicity. Suitable surface modification of MSNs from negative to positive is a prerequisite parameter for conjugation of biological molecules (siRNA or DNA).

50

For ligand conjugation, the negative surface of the MSNs was

made positive with polymer coating of polyethyleneimine (PEI). Similar type of PEI coating 7



were used for ligand conjugation in MSNs by Ngamcherdtrakul et al. 50 The surface morphology of MSNs as studied by AFM and SEM depicted smooth and rounded structure (~161 nm ± 3 nm (taken from 50 measurements) in average diameter and the internal structure as observed by TEM showed rounded porous structure of MSNs (Figure 1a, b, c). The electron diffraction rings of selected area electron diffraction (SAED) pattern of synthesized MSNs confirmed uniform and amorphous state of the MSNs (Figure 1d). The elemental composition of Si and O of MSNs determined by energy dispersive analysis of X- rays (EDAX) spectrum showed sharp peak of silica and oxygen (Figure 1e). Further the amorphous nature of MSNs has been confirmed through X-ray diffraction (XRD) which illustrated a crystalline peak around ~ 22.8° depicting the presence of silica with no other impurities (Figure 1f). Furthermore, MSNs material characterization of the pore structure, specific surface area, and pore volume of MSNs were determined by the by Brunauer Emmett Teller (BET) and Barret Joyner Halenda (BJH) methods using nitrogen adsorption–desorption isotherms. An illustration of the N2 adsorption–desorption isotherms of MSNs is shown in (Figure 1g) which displayed a type IV isotherm, characteristic of mesoporous materials, featuring a narrow step due to capillary condensation of N2 within the primary mesopores according to IUPAC classification.51 Figure 1h, exhibited the BJH adsorption average pore diameter (4V/A): 38.857 Å (~ 3.86 nm) with a peak around 30 Å (~ 3 nm) and the mesostructure of MSNs were very uniform with the BET surface area: 780.6159 m2 g-1, and a pore volume at P/Po = 0.97 of 0.72 cm³ g-1. Various research groups engaged in synthesis of MSNs have also observed similar type IV isotherm patterns and trends related to surface area, pore volume and average diameter. 9,52-54

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In vitro release profile of individual drug from combined drug formulations demonstrated biphasic release pattern and the difference in release pattern may be attributed to the hydrophobic nature of both the drugs in formulations (Figure S1). Using Differential Scanning Calorimetry (DSC), the nature of encapsulated drug in the drug loaded MSNs were studied. The analysis was done in native drug and drug loaded MSNs. The absence of drug peak in drug loaded nanoformulations illustrated the amorphous or disorder crystalline phase or solid solution state of drugs (Figure S2). Fourier transmission infrared (FTIR) spectra of void-MSNs, showed two strong bands at ~3435 cm−1 and ~1635 cm−1, which are due to the adsorbed water and assigned to the O–H stretching vibration of water molecules. The other bands at ~1091 cm−1 and ~ 966 cm−1 are assigned to the Si–O and Si–OH vibrations, respectively. The band at 797 cm−1 is assigned for the formation of condensed (Si-O-Si) silica network.55 Further, we have studied the characteristics peaks of individual drugs in combination and analysis revealed the presence of characteristics peaks of individual native drugs in combined drug loaded nanoformulation, indicating the efficient loading of drug in the formulations (Figure S3).

Intracellular uptake of FAM labeled siRNA and 6-coumarin-MSNs in A549 cells The fluorescence property of fluorescein amidite (FAM) labeled si-RNA was explored to know the cellular localization of si-RNA in the cytoplasm. The cationic complex (FAM siRNAPEI-MSNs) facilitates the internalization of siRNA and its localization was visualized with confocal microscopy. A549 cells were incubated with FAM labeled si-RNA (20 nM) either in native or equivalent concentration in MSNs cationic complex for 6 h. There was an increased 9



cellular uptake of FAM–si-RNA in MSN cationic complex than that of native at observed time period (Figure 2a). In our earlier publication intracellular uptake of non-fluorescent drug was studied using photoluminescent 6-coumarin. 56 With the help of intrinsic fluorescent property of 6-coumarin, the comparative analysis of intracellular uptake of native 6-coumarin and 6coumarin loaded-MSNs were observed in A549 cells for a period of 6 h by flow cytometry. Results demonstrated augmented cellular uptake of 6-coumarin loaded MSNs than that of native 6-coumarin. The mean fluorescence intensity (MFI) obtained from the study depicted ~2.2 times enhanced MFI in 6-coumarin loaded MSNs as compared to native 6-coumarin, suggesting better internalization of MSNs by the cancer cells (Figure 2b, c).

In vitro cytotoxic effect of void and drug loaded loaded MSNs

Initially, we have evaluated the cytotoxic effect of void MSNs in normal cell line (HEK293) and cancerous cell line (A549) by MTT assay and found up to ~ 1 mg/ml of void MSNs is not toxic to both the cell lines (Figure S4a, b). Therapeutic efficiency of drug loaded nanosystems is primarily dependant on uptake of nanoparticle, its intracellular distribution, and more importantly the amount of drug that is released from the internalized nanoparticles inside the cell

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. Anticancer effects of different formulations were analyzed in both the cell lines

(HEK293 and A549 cells) by MTT assay. All the single drug loaded MSN formulations were capable of inducing pronounced cell inhibitory effect in non-malignant HEK293 at a very low concentration as observed from IC50, illustrating its susceptibility to the treatments (Figure S4c, Table S2). Whereas, in malignant A549 cells, significant dose -dependent inhibitory effects were observed (Figure 3a, Table 1). Remarkable progress has been achieved using MSNs as combinatorial delivery systems. MSNs are highly advantageous for such co-delivery systems 10


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because of their well-established surface chemistry and porous structure. With combination therapies, co-delivery agents are precisely targeted to the cells which in turn illustrate enhanced therapeutic effects making it a strategy of increasing interest for cancer therapy. In our study it is discernible that the combined drug loaded MSNs induce pronounced cell inhibitory effect on A549 cells (Figure 3b, c). Addition of proteasomic inhibitory drug CAR to the formulations has drastically reduced the IC50 values in combined formulations compared to single formulations (Table 2). Further, it was found that the si-RNA complexed with MSNs loaded combined drugs have shown augmented cytotoxicity as compared to its combined drug loaded MSNs and their combined native counterparts (Figure 3b, c). The IC50 values of all formulations (combined native, combined drug loaded nanoformulations, si-RNA complexed combined drug loaded nanoformulations) are presented in (Table 2). The results revealed that the si-RNA complexed combinational nanoformulations have lower IC50 as compared to its respective uncomplexed nanoformulations and combined native counterparts. Similar type of results was also reported by Zhou et al, illustrating superior cytotoxic activity with co-delivery of DOX and Bcl-2 si-RNA in DOX@MSNs-PPPFA/Bcl-2 si-RNA nanocomplexes in MDA-MB-231 cells compared to DOX delivery alone. Functionalization with ligand increases the internalization of nanoparticles via overexpressed receptor mediated endocytosis which led to improved cytotoxic effect.

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In

another study, Meng et al used DOX loaded (P-g)-si-RNA functionalized DOX-MSNs for accomplishing better cytotoxic activity in drug resistant squamous carcinoma cell line KB-V1. The result demonstrated increased intracellular uptake and intranuclear drug concentration along with knocking down of drug exporter compared to free DOX. 55 In another study, Taratula et al. developed MSNs loaded with anticancer drugs, targeted with LH-RH and complexed with different si-RNAs for lung cancer through local delivery by inhalation in an animal model. The 11



loaded drugs (doxorubicin and cisplatin) were effectively delivered with two types of si-RNA targeted to MRP1 and Bcl2 mRNA for the suppression of pump and nonpump cellular resistance in non-small cell lung carcinoma, respectively.59 Various clinical studies have demonstrated that combination of anticancer drugs resulted in a more efficient tumor regression compared to drug alone.

60,61

Nanocarrier containing cytotoxic drugs with independent mechanisms of action can

give desirable nature of cancer treatment. 62,63

Measurement of mitochondrial membrane potential (∆Ψm) loss

In cellular apoptosis process, mitochondria play a very vital role. The key feature to mark the initiation of apoptotic event is the depolarization of the mitochondrial membrane potential leading to membrane potential loss.64,65 The mitochondrial membrane potential (MMP) loss was evaluated in different nanoformulations by flow cytometry with Mito Tracker Red CMXRos staining. Result depicted that MMP loss was higher in case of cells treated with combined drug loaded MSNs as compared to their respective combined native counterparts. However, exposure of cells to si-RNA functionalized drug formulations significantly decreased the MMP as compared to all other treatments in A549 cells (Figure 4a, b). The corresponding MFI values obtained from treatments showed significant mitochondrial membrane potential loss (Figure 4c, d).

The higher loss of MMP activated the apoptotic pathways in A549 cells. In this context we have evaluated the expression of apoptotic proteins Bax and Bcl-2 in different treatment groups. Results indicated that there was enhanced downregulation of proapoptotic Bcl-2 protein and upregulation of Bax protein in si-RNA complexed combined drug loaded MSNs as 12


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compared to combined drug loaded MSNs counterpart and combined native drugs of both the formulations (Figure 4e, f). Therefore, the results of the present investigation suggested that simultaneous delivery of si-RNA and drug by cationic MSNs enhanced the apoptotic process by increasing the cytotoxicity in cancer cells.

Suppression of survivin si-RNA and augmented apoptosis

Survivin is an essential component of multiple functional protein complexes in cancer cells. Survivin, an inhibitor of apoptosis protein is highly expressed in most of the cancers and is associated with tumor recurrence, apoptosis, cellular stress response, regulation of cell migration, chemoresistance and metastasis leading to low patient survivility.66 The si-RNA based therapeutic approach has been regarded as a powerful gene delivery technology for wide range of cancers by enabling post-transcriptional gene silencing which suppresses the activity of the targeted mRNA, hence making anti-survivin therapy an attractive mode for cancer treatment. Herein, the silencing ability of the complexed survivin si-RNAs delivered along with drug loaded MSNs was evaluated using quantitative RT-PCR. Results suggested that, the silencing efficiency of si-RNA complexed combined drug loaded MSNs was higher than that of combined drug loaded MSNs and combined native drugs (Figure 5a, b). Significant gene silencing effect in si-RNA complexed nanoparticles can be attributed to their higher cellular uptake. Further the gene suppression level was studied by western blot analysis using different formulations which corroborated the downregulation of survivin protein activity (Figure 5c, d). Numerous researches have stated the therapeutic efficacy of survivin based strategy. Mattheolabakis et al developed lipid-modified platinum compounds with survivin-si-RNA for in vitro and in vivo evaluation in lung cancer. These compounds illustrated strongest tumor growth inhibition in 13



combination of survivin si-RNA on a xenograft cancer model compared to free cisplatin drug.67 Steinbacher and Landry reported remarkable cytotoxicity by Bcl-2-si-RNA- doxorubicin loaded MSNs compared to free drug.68 Xu et al, developed N-β-maleimidopropionic acid hydrazide and PEI based polymeric nanoparticle for co-delivery of doxorubicin and survivin si-RNA for the treatment of metastatic lung cancers by pulmonary delivery which resulted in preferential accumulation of doxorubicin and si-RNA in the lungs in the B16F10 tumor-bearing mice models with enhanced antitumor efficacy as compared with the monodelivery of doxorubicin or survivin si-RNA.69 The delivered anticancer drugs and si-RNA illustrated specific activity that led to the cell death induction and inhibition of targeted mRNA substantiating the enhanced cytotoxicity of anticancer drugs. 59

The study of apoptotic event executed by combined native drugs, combined drug loaded MSNs and si-RNA complexed combined drug loaded MSNs in A549 cells by flow cytometry revealed that, si-RNA conjugated combined drug loaded MSNs has resulted in higher apoptotic cell death as compared to combined drug loaded MSNs than its respective combined native counterparts in the studied formulations (Figure 5e, f), whereas (Figure 5g, h) represents number of respective apoptotic cell death for the nanoformulations. It is worth mentioning that; exposure of cells to combination of drugs significantly increased the number of apoptotic cells as compared to all other treatments. Thus, anticancer effect of the proposed MSNs that was capable of co-delivering anticancer drugs as (cell death inducers) and survivin-si-RNA as (suppressors of survivin) has contributed to higher cytotoxic activity. Both the formulations induced greater cytotoxicity in A549 cells, following enhanced uptake along with dual modality of action of gene silencing as well as combinational drug effect. Moreover, addition of CAR (anticancer drug 14


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acting like selective irreversible proteasome inhibitor) to the formulations significantly reduced the IC50 values compared to single drug loaded MSNs. In addition, survivin si-RNA complexed nanoformulations further enhanced the gene suppression effect which led to superior cytotoxicity compared to native drug alone. The comparative assessment between the two formulations revealed better efficacy for MSNs (ETO +CAR)-si in vitro. However further studies will be continued for the evaluation of anticancer property of co-delivery of combined drugs along with gene silencing strategy behavior in lung cancer model for validation of the targeted therapy in the long run.

Conclusion In summary, this study reported the development of MSNs based drug delivery for lung cancer therapy. The physicochemical characterization of synthesized MSNs has been systematically investigated and to that highly hydrophobic drugs and proteasome inhibitor carfilzomib were successfully entrapped. These drug loaded MSN formulations illustrated good dispersibility, sustained drug release activity, biocompatibility and low toxicity. High levels of survivin expression have been associated in lung cancers, different approaches have been aimed to curtail its expression. Amongst them currently, RNAi therapeutic based strategy has been intended for its successful implementation. In the present study survivin si-RNA complexed combined drug loaded MSNs were assessed for its cytotoxic effect in A549 cells. Owing to better cellular uptake along with suppression of survivin expression both formulated combined drug loaded nanoparticles exhibited superior in vitro cytotoxicity. Mitochondrial depolarization and apoptosis studies of these two different combined drug loaded MSNs demonstrated pronounced apoptosis effect. The difference observed in dose exerted cytotoxicity between (ETO + CAR) and (DOX + 15



CAR) formulations in lung cancer cells is greatly dependent on the combined drug interaction between themselves as well as in the cells. In this regard MSNs mediated RNAi based therapeutic strategy may emerge as better approach for lung cancer therapy.

Acknowledgements This work was supported by a financial grant [SR/WOS-A/LS-524/2013 (G)] to FD in the form of Women Scientist Scheme (WOS-A) from the Department of Science and Technology, Government of India. The authors gratefully acknowledge Dr Dipti P. Das of Institute of Minerals and Materials Technology, Bhubaneswar for her help in conducting BET measurement and Mr Ajit Kumar Das for his help in TEM images. The help of Mr. Bhabani Shankar Sahoo during the confocal experiments, Ms Somalisa Behera for western blot experiments and Mr. Priyadarshi Roy for taking images in AFM at Institute of Life Sciences are thankfully acknowledged.

Supporting Information Available: [Materials and Methods, detailed synthetic procedures, physico-chemical characterization like DSC and FTIR analysis, loading and entrapment efficiency of different drugs by HPLC, in vitro release kinetics study, adsorption of si-RNA on to PEI-MSNs and absorption analysis by agarose gel retardation assay, cell culture, qualitative cellular uptake, cytotoxicity assessment, tables of physicochemical characterization of different nanoformulations and its in vitro cytotoxicity profile, mitochondrial membrane potential study, apoptosis assay,

effects of si-RNA on survivin expression/apoptotic protein and gene

expression, statistical analysis.]

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References

(1)

Siegel, R.L.; Miller, K.; Jemal, A. Cancer Statistics, 2017. CA Cancer J. Clin. 2017, 67, 7-30.

(2)

Baker, S.; Dahele, M.; Lagerwaard, F.J.; Senan, S. A critical review of recent developments in radiotherapy for non-small cell lung cancer. Radiation Oncology 2016, 11, 1-14.

(3)

Clegg, A.; Scott, D.; Hewitson, P.; Sidhu, M.; Waugh, N. Clinical and cost effectiveness of paclitaxel, docetaxel, gemcitabine, and vinorelbine in non-small cell lung cancer: a systematic review. Thorax 2002 57, 20-28.

(4)

Fuld, A.D.; Dragnev, K.; Rigas, J.R. Pemetrexed in advanced non-small-cell lung cancer. Expert Opin. Pharmacother. 2010, 11, 1387-1402.

(5)

Wang, C.; Li, Z.; Cao, D.; Zhao, Y-L.; Gaines, J.W.; Bozdemir, O.A.; Ambrogio, M.W.; Frasconi, M.; Botros, Y.Y.; Zink, J.I.; Stoddart, J.F. Stimulated release of size-selected cargos in succession from mesoporous silica nanoparticles. Angew. Chem. Int. Ed. Engl. 2012, 51, 5460-5465.

(6)

Zhou, S.; Sha, H;, Liu, B; Du, X. Integration of simultaneous and cascade release of two drugs into smart single nanovehicles based on DNA-gated mesoporous silica nanoparticles. Chem. Sci. 2014, 5, 4424-4433.

17



(7)

Eytan, G, D. Mechanism of multidrug resistance in relation to passive membrane permeation. Biomed. Pharmacother. 2005, 59, 90-97.

(8)

Szakács, G.; Paterson, J.; Ludwig, J.A. Booth-Genthe C, Gottesman MM Targeting multidrug resistance in cancer. Nat. Rev. Drug Discov. 2006, 5, 219-234.

(9)

Chen, A.M.; Zhang, M.; Wei, D.; Stueber, D.; Taratula, O.; Minko, T.; He, H. Codelivery of doxorubicin and Bcl-2 siRNA by mesoporous silica nanoparticles enhances the efficacy of chemotherapy in multidrug-resistant cancer cells. Small 2009, 5, 26732677.

(10)

Hideshima, T.; Richardson, P.; Chauhan, D.; Palombella, V.J.; Elliott, P.J.; Adams, J.; Anderson, K.C. The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells. Cancer Res. 2001, 61, 3071-3076.

(11)

Landis-Piwowar, K.R.; Milacic, V.; Chen, D.; Yang, H.; Zhao, Y.; Chan, T.H.; Yan, B.; Dou, Q.P. The proteasome as a potential target for novel anticancer drugs and chemosensitizers. Drug Resistance Updates 2006, 9 , 263-273.

(12)

Mani, A.; Gelmann, E. The ubiquitin-proteasome pathway and its role in cancer. J. Clin. Oncol. 2005, 23, 4776-4789.

(13)

Shen, M.; Schmitt, S.; Buac, D. Dou, Q.P. Targeting the ubiquitin–proteasome system for cancer therapy. Expert Opin. Ther. Targets. 2013, 17, 1091-1108.

18


Page 18 of 36

Page 19 of 36 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


(14)

Demo, S.D.; Kirk, C.; Aujay, M.A.; Buchholz, T.J.; Dajee, M.; Ho, M.N.; Jiang, J.; Laidig, G.J.; Lewis, E.R.; Parlati, F.; Shenk, K.D.; Smyth, M.S.; Sun, C.M.; Vallone, M.K.; Woo, T.M.; Molineaux, C.J.; Bennett, M.K. Antitumor activity of PR-171, a novel irreversible inhibitor of the proteasome. Cancer Res. 2007, 67, 6383-6391.

(15)

Zhang, J.; Wu, P.; Hu. Y. Clinical and marketed proteasome inhibitors for cancer treatment. Curr. Med. Chem. 2013, 20, 2537-2551.

(16)

Baker, A.F.; Hanke, N.; Sands, B.J.; Carbajal, L.; Anderl, J.L.; Garland, L. L. Carfilzomib demonstrates broad anti-tumor activity in pre-clinical non-small cell and small cell lung cancer models. J. Expt. Clinical Cancer Res. 2014, 33, 1-12.

(17)

Kuhn, D.J.; Chen, Q.; Voorhees, P.M.; Strader, J.S.; Shenk, K.D.; Sun, C.M.; Demo, S.D.; Bennett, M.K.; van Leeuwen, F.W.; Chanan-Khan, A.A.; Orlowski, R.Z. Potent activity of carfilzomib, a novel, irreversible inhibitor of the ubiquitinproteasome pathway, against preclinical models of multiple myeloma. Blood 2007, 110, 3281-3290.

(18)

Li, F.; Ackermann, E.; Bennett, C.F.; Rothermel, A.L.; Plescia, J.; Tognin, S.; Villa, A.; Marchisio, P.C.; Altieri, D.C. Pleiotropic cell-division defects and apoptosis induced by interference with survivin function. Nat. Cell Biol. 1999, 1, 461-466.

(19)

Pennati, M.; Binda, M.; Colella, G.; Zoppe, M.; Folini, M.; Vignati, S.; Valentini, A.; Citti, L.; De Cesare, M.; Pratesi, G.; Giacca, M.; Daidone, M.G.;

Zaffaroni, N.

Ribozyme-mediated inhibition of survivin expression increases spontaneous and drug-

19



induced apoptosis and decreases the tumorigenic potential of human prostate cancer cells. Oncogene 2004, 23, 386-394.

(20)

Shen, C.; Buck, A.; Polat, B.; Schmid-Kotsas, A.; Matuschek, C.; Gross, H.J.; Bachem, M.; Reske, S.N. Triplex-forming oligodeoxynucleotides targeting survivin inhibit proliferation and induce apoptosis of human lung carcinoma cells. Cancer Gene Ther. 2003, 10, 403-410.

(21)

Hannon, G.J.; Rossi, J. Unlocking the potential of the human genome with RNA interference. Nature 2004, 431, 371-378.

(22)

Menendez, J.A.; Vellon. L.; Mehmi, I.; Oza, B.P.; Ropero, S.; Colomer, R.; Lupu, R. Inhibition of fatty acid synthase (FAS) suppresses HER2/neu (erbB-2) oncogene overexpression in cancer cells. Proc. Natl. Acad. Sci. U S A 2004, 101, 10715-10720.

(23)

Whitehead, K.A.; Langer, R.; Anderson, DG. Knocking down barriers: advances in siRNA delivery. Nat Rev. Drug Discov. 2009, 8, 129-138.

(24)

Liu, J.; Li, C.; Li, F. Fluorescence turn-on chemodosimeter-functionalized mesoporous silica nanoparticles and their application in cell imaging. J. Mater. Chem. 2011, 21, 7175-7181.

(25)

Ariga, K.; Ji, Q.; Mori, T.; Naito, M.; Yamauchi, Y.; Abe, H.; Hill, J.P. Enzyme nanoarchitectonics: organization and device application. Chem. Soc. Rev. 2013, 42, 63226345.

20


Page 20 of 36

Page 21 of 36 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


(26)

Ehlert, N.; Mueller, P.; Stieve, M.; Lenarz, T.; Behrens, P. Mesoporous silica films as a novel biomaterial: applications in the middle ear. Chem. Soc. Rev. 2013, 42, 3847-3861.

(27)

Salinas, A. J., Esbrit P, Vallet-Regí M. A tissue engineering approach based on the use of bioceramics for bone repair. Biomater. Sci. 2013, 1, 40-50.

(28)

Lee, J.E.; Lee. N.; Kim, T.; Kim, J.; Hyeon, T. Multifunctional mesoporous silica nanocomposite nanoparticles for theranostic applications. Acc. Chem. Res. 2011, 44, 893902.

(29)

Liong, M.; Lu. J.; Kovochich, M.; Xia, T.; Ruehm, S.G.; Nel, A.E.; Tamanoi, F.; Zink, J.I. Multifunctional inorganic nanoparticles for imaging, targeting, and drug delivery. ACS Nano 2008, 2, 889-896.

(30)

Li, Z.; Barnes, J.; Bosoy, A.; Stoddart, J. F.; Zink, J.I. Mesoporous silica nanoparticles in biomedical applications. Chem. Soc.Rev. 2012, 41, 2590-2605.

(31)

Maher S, Kumeria T, Wang Y, Kaur G, Fathalla D, Fetih G, Santos A, Habib F, Evdokiou A, Losic D. From The Mine to Cancer Therapy: Natural and Biodegradable Theranostic

Silicon

Nanocarriers

from

Diatoms

for

Sustained

Delivery

of

Chemotherapeutics. Adv. Healthc. Mater. 2016, 5, 2667-2678.

(32)

Vivero-Escoto, J.L.; Slowing, I.; Trewyn, B.G.; Lin, V.S. Mesoporous silica nanoparticles for intracellular controlled drug delivery. Small 2010, 6, 1952-1967.

21



(33)

Knežević,N.Z.; Kaluđerović, G.N. Silicon-based nanotheranostics. Nanoscale. 2017, 9, 12821-12829.

(34)

Aznar, E.; Mondragon, L.; Ros-Lis, J.V.; Sancenón, F.; Marcos, M.D.; Martínez-Máñez, R.; Soto, J.; Pérez-Payá, E.; Amorós, P. Finely tuned tempera¬ture-controlled cargo release using paraffin-capped mesoporous silica nanoparticles. Angew. Chem .Int. Ed. Engl. 2011, 50, 11172-11175.

(35)

Schlossbauer, A.; Warncke, S.; Gramlich, P.M.; Kecht, J.; Manetto, A.; Carell, T.; Bein, T. A programmable DNA-based molecular valve for colloidal mesoporous silica. . Angew. Chem. Int. Ed. Engl. 2010, 49, 4734-4737.

(36)

Yang, X.J.;

Liu, X.; Liu, Z.; Pu, F.; Ren, J.; Qu, X. Near-infrared light-triggered,

targeted drug delivery to cancer cells by aptamer gated nanovehicles. Adv. Mater. 2012, 24, 2890-2895.

(37)

Bernardos, A.; Aznar, E.; Marcos, M.D.; Martínez-Máñez, R.; Sancenón, F.; Soto, J.; Barat, J.M.; Amorós, P. Enzyme-responsive controlled release using mesoporous silica supports capped with lactose. Angew. Chem. Int. Ed. Engl. 2009, 48, 5884-5887.

(38)

Schlossbauer, A.; Kecht, J.; Bein, T. Biotin-avidin as a protease-responsive cap system for controlled guest release from colloidal mesoporous silica. Angew. Chem. Int. Ed. Engl. 2009, 48, 3092-3095.

22


Page 22 of 36

Page 23 of 36 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


(39)

Hu, C.; Yu, L.; Zheng, Z.; Wang, J.; Liu, Y.; Jiang, Y.; Tong, G.; Zhou, Y.; Wang, X. Tannin as a gatekeeper of pH-responsive mesoporous silica nanoparticles for drug delivery. RSC Adv. 2015, 5, 85436-85441.

(40)

Giri, S.; Trewyn, B.; Stellmaker, M.P.; Lin, V.S. Stimuli-responsive controlled-release delivery system based on mesoporous silica nanorods capped with magnetic nanoparticles. Angew. Chem. Int. Ed. Engl. 2005, 44, 5038-5044.

(41)

Kim, H.; Kim, S.; Park, C.; Lee, H.; Park, H.J.; Kim, C. Glutathione-induced intracellular release of guests from mesoporous silica nanocontainers with cyclodextrin gatekeepers. Adv. Mater. 2010, 22, 4280-4283.

(42)

Jurkić, L.M.; Cepanec, I.; Pavelić, S.K . Pavelić K. Biological and therapeutic effects of ortho-silicic acid and some ortho-silicic acid-releasing compounds: New perspectives for therapy. Nutr. Metab (Lond) 2013, 10, 1-12

(43)

Bivas-Benita, M.; Romeijn, S.; Junginger, H.E.; Borchard, G. PLGA-PEI nanoparticles for gene delivery to pulmonary epithelium. Eur. J. Pharm. Biopharm. 2004, 58, 1-6.

(44)

Godbey,W.T.;

Wu,

K.;

Hirasaki,

G.J.;

Mikos,

A.G.

Improved

packing

of

poly(ethylenimine)/DNA complexes increases transfection efficiency. Gene Ther. 1999, 6 , 1380-1388.

(45)

Zhang, W.; Shen, J.; Su, H.; Mu, G.; Sun, J.H.; Tan, C.P.; Liang, X.J.; Ji, L.N.; Mao, Z.W. Co-Delivery of Cisplatin Prodrug and Chlorin e6 by Mesoporous Silica

23



Nanoparticles for Chemo-Photodynamic Combination Therapy to Combat Drug Resistance. ACS Appl. Mater. Interfaces 2016, 8, 13332-13340.

(46)

Lu, H.Y.; Chang, Y.; Fan, N.C.; Wang, L.S.; Lai, N.C.; Yang, C.M.; Wu, L.C.; Ho, J.A. Synergism through combination of chemotherapy and oxidative stress-induced autophagy in A549 lung cancer cells using redox-responsive nanohybrids: A new strategy for cancer therapy. Biomaterials. 2015, 42, 30-41.

(47)

Falleni, M.; Pellegrini, C.; Marchetti, A.; Oprandi, B.; Buttitta, F.; Barassi, F.; Santambrogio, L.; Coggi, G.; Bosari, S. Survivin gene expression in early-stage nonsmall cell lung cancer. J. Pathol. 2003, 200, 620-626.

(48)

Zheng, H.; Tai. C.W.; Su, J.; Zou, X.; Gao, F.Ultra-small mesoporous silica nanoparticles as efficient carriers for pH responsive releases of anti-cancer drugs. Dalton Trans. 2015, 44, 20186-20192.

(49)

Zhou S, W. D., Yin X, Jin X, Zhang X, Zheng S, Wang C, Liu Y. Intracellular pHresponsive and rituximab-conjugated mesoporous silica nanoparticles for targeted drug delivery to lymphoma B cells. J. Exp. Clin. Cancer Res. 2017, 36, 1-14.

(50)

Ngamcherdtrakul, W.; Morry, J.; Gu, S.; Castro, D.J.; Goodyear, S.M.; Sangvanich, T.; Reda, M.M.; Lee, R.; Mihelic, S.A.; Beckman, B.L.; Hu, Z.; Gray, J.W.; Yantasee, W. Cationic polymer modified mesoporous silica nanoparticles for targeted siRNA delivery to HER2+ breast cancer. Adv. Funct. Mater. 2015, 25, 2646-2659

24


Page 24 of 36

Page 25 of 36 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


(51)

Xiao, X.; Liu, Y.; Guo, M.; Fei, W.; Zheng, H.; Zhang, R.; Zhang, Y.; Wei, Y.; Zheng, G.; Li, F. pH-triggered sustained release of arsenic trioxide by polyacrylic acid capped mesoporous silica nanoparticles for solid tumor treatment in vitro and in vivo. J. Biomater. Appl 2016, 31, 23-35.

(52)

Al-Attar, L.; Dyer, A.; Harjula, R. Uptake of radionuclides on microporous and layered ion exchange materials. J. Mater. Chem. 2003, 13, 2963-2968.

(53)

Lin Y-C, L. L.-Y., Gao M-Y, Fang Y-P. Mesoporous silica nanoparticles synthesized from liquid crystal display manufacturing extracts as a potential candidate for a drug delivery carrier: evaluation of their safety and biocompatibility. Intert. J. Nanomed. 2013, 8, 3833-3842.

(54)

Sundarraj, S.; Thangama, R.; Sujitha, M.V.; Vimala, K.; Kannan, S. Ligand-conjugated mesoporous silica nanorattles based on enzyme targeted prodrug delivery system for effective lung cancer therapy. Toxicol. Appl. Pharmaco. 2014, 275, 232-243.

(55)

Meng, H.; Liong, M.; Xia, T.; Li, Z.; Ji, Z.; Zink, J.I.; Nel, A.E. Engineered design of mesoporous silica nanoparticles to deliver doxorubicin and P-glycoprotein siRNA to overcome drug resistance in a cancer cell line. ACS Nano 2010, 4, 4539-4550.

(56)

Dilnawaz, F.; Singh, A.; Mewar, S.; Sharma, U.; Jagannathan, N.R.; Sahoo, S.K. The transport of non-surfactant based paclitaxel loaded magnetic nanoparticles across the blood brain barrier in a rat model. Biomaterials, 2012, 33, 2936-2951.

25



(57)

Panyam, J.; Labhasetwar, V. Biodegradable nanoparticles for drug and gene delivery to cells and tissue. Adv. Drug Deliv. Rev. 2003, 55, 329-347.

(58)

Zhou, X.; Chen, L.; Nie, W.;Wang, W.; Qin, M.; Mo, X.; Wang, H.; He, C. DualResponsive Mesoporous Silica Nanoparticles Mediated Codelivery of Doxorubicin and Bcl-2 SiRNA for Targeted Treatment of Breast Cancer. J. Phys. Chem. C 2016, 120, 22375-22387.

(59)

Taratula, O.; Garbujenko, O.; Chen, A.M.; Minko, T. Innovative strategy for treatment of lung cancer: targeted nanotechnology-based inhalation co-delivery of anticancer drugs and siRNA. J. Drug. Target. 2011, 19, 900-914.

(60)

Chen, G.; Huynh, M.; Fehrenbacher, L.; West, H.; LaraJr, P.N.; Yavorkovsky, L.L.; Russin, M.;

Goldstein, D.; Gandara, D.; Lau, D. Phase II Trial of Irinotecan and

Carboplatin for Extensive or Relapsed Small-Cell Lung Cancer, J. Clin. Oncol, 2009, 27, 1401–1404

(61)

Pinmai, K.; Chunlaratthanabhorn, S.; Ngamkitidechakul, C.;Soonthornchareon, N.; Hahnvajanawong, C. Synergistic growth inhibitory effects of Phyllanthus emblica and Terminalia bellerica extracts with conventional cytotoxic agents: Doxorubicin and cisplatin against human hepatocellular carcinoma and lung cancer cells. World J. Gastroenterol 2008, 14, 1491-1497.

26


Page 26 of 36

Page 27 of 36 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


(62)

Acharya, S.; Sahoo, S. K. Exploitation of redox discrepancy in leukemia cells by a reactive oxygen species nanoscavenger for inducing cytotoxicity in imatinib resistant cells. J. Colloid Inter. Sci. 2016, 467, 180-191.

(63)

Hu, C.M.; Zhang, L. Nanoparticle-based combination therapy toward overcoming drug resistance in cancer. Biochem. Pharmacol. 2012, 83, 1104-1111.

(64)

Pendergrass, W.; Wolf, N.; Poot, M. Efficacy of MitoTracker Green and CMXrosamine to measure changes in mitochondrial membrane potentials in living cells and tissues. Cytometry A 2004, 61, 162-169.

(65)

Sareen, D.; van, Ginkel. P., Takach, J.C.; Mohiuddin, A.; Darjatmoko, S.R.; Albert, D.M.; Polans, A.S. Mitochondria as the Primary Target of Resveratrol-Induced Apoptosis in Human Retinoblastoma Cells. Investig. Opthal. Visual Sci. 2006, 47, 3708-3716.

(66)

Groner, B.; Weiss, A. Targeting Survivin in Cancer: Novel Drug Development Approaches. BioDrugs. 2014, 28, 27-39.

(67)

Mattheolabakis, G.; Ling, D.; Ahmad, G.; Amiji, M. Enhanced Anti-Tumor Efficacy of Lipid-Modified Platinum Derivatives in Combination with SurvivinSilencing siRNA in Resistant Non-Small Cell Lung Cancer. Pharmaceu. Res. 2016, 33, 2943-2953.

(68)

Steinbacher, J.L.; Landry, C. Adsorption and Release of siRNA from Porous Silica. Langmuir, 2014, 30, 4396-4405.

27



(69)

Xu, C.; Tian, H.; Wang, P.1.; Wang, Y.; Chen, X. The suppression of metastatic lung cancer by pulmonary administration of polymer nanoparticles for co-delivery of doxorubicin and Survivin siRNA. Biomater. Sci. 2016, 4, 1646-1654.

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Page 28 of 36

Page 29 of 36

a

b

c

d

e

f

g

800

600

h

700

0.10

Volume Adsorbed (cm /g, STP)

500

600 500

0.08

400

400 300 200

300

°

200

100

dV/dD(cm³/gA)

3

Intensity (a.u)

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


0.06

0.04

0.02

100 0

10

20

30

40

2 theta (degrees)

50

60

70

0.00

0.0

0.2

0.4

0.6

0.8

1.0

Relative Pressure (P/P0)

0

20

40

60

80

100

° Pore Diameter (A)

120

140

Figure 1: Physico-chemical characterization of MSNs. (a) Atomic force microscopy image of MSNs. (b) Scanning electron microscopy image of MSNs. (c) Transmission electron micrograph of MSNs. (d) Selected area diffraction pattern (SADP) of MSNs. (e) Energy dispersive X-ray analysis (EDX) of MSNs. (f) Powder wide angle X-Ray diffraction pattern of MSNs. (g) Nitrogen adsorption/desorption isotherms of MSN. (h) Pore size distribution of MSNs at standard temperature and pressure (STP). 29


160


a

Merged

MSNs

NAT

CONTROL

DAPI

c

b CONTROL

Counts

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

FAM-siRNA

Page 30 of 36

NAT (6-COUMARIN)

***

(6-COUMARIN)-MSNs

FITC-A Figure 2: Cellular uptake study (a) Intracellular uptake of FAM labeled si-RNA (NAT and MSN formulations) at 6 h in A549 cells by confocal microscopy. Experiments were done in triplicate and a representative figure has been provided. (b) Cellular uptake for 6 h of native 6-Coumarin and 6Coumarin MSNs (30 ng/ml) in A549 cells by flow cytometer. Experiments were done in triplicate and a representative figure has been provided. (c) Mean fluorescence intensity (MFI) obtained from the flow cytometry analysis of cellular uptake. (n=3: data as mean ± S.D). ***p < 0.0001, NAT (6-COUMARIN) vs (6- COUMARIN)-MSNs 30


Page 31 of 36 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


a

a

b

c

Figure 3: Dose dependent cytotoxicity study in single formulation of (a) NAT-CAR ( ) CAR-MSNs ( ) NAT-ETO ( ) ETO-MSNs ( ) NAT-DOC ( ) and DOC-MSNs (

) in A549 cells after 3 days of drug

treatment by MTT assay. (b) Dose dependent cytotoxicity study of combined formulations of NAT (ETO + CAR) (

) NAT (ETO + CAR)-si ( ), MSNs (ETO + CAR) (

NAT (DOC + CAR) ( CAR)-si (

) and MSNs (ETO + CAR)-si (

) and NAT (DOC + CAR)-si ( ), MSNs (DOC + CAR) (

). (c)

), MSNs (DOC +

) in A549 cell line after 3 days of drug treatment by MTT assay. (n=3: data as mean ±

S.E.M). ***p < 0.0001, NAT drug vs drug loaded MSNs or combined drug MSNs vs combined drug MSNs-si. 31


a

b CONTROL

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


c

CONTROL

###

***

***

***

NAT (ETO+ CAR)

NAT (DOC+ CAR)

e

1

3

2

f

4

2

3

4

0.961

0.981

1

1.15

β actin MSNs (DOC+CAR)

β actin Bax/ β actin

MSNs (ETO +CAR)-si

1

Bax

Bax MSNs (ETO + CAR)

Page 32 of 36 ***

d

###

MSNs (DOC+CAR)-si

0.779

0.842

1

2

0.952

3

1.04

Bax/ β actin

1

4

Bcl 2

Bcl 2

β actin

β actin

Bcl2/ β actin

1.09

1.04

0.924

0.775

Bcl2/ β actin

2

0.9

0.821

3

0.781

Figure 4: Flow cytometry study of mitochondrial membrane potential by Mito Tracker®Red CMXRos in A549 cells. (a) Histogram plots of NAT (ETO + CAR), MSNs (ETO + CAR), MSNs (ETO + CAR) –si. (b) Histogram plots of NAT drugs (DOC + CAR), MSNs (DOC + CAR), MSNs (DOC + CAR)-si. (c) and (d) Respective mean fluorescent intensity (MFI) obtained from above histogram plots. (n = 3: data as mean ± S.D). ***p < 0.0001, combined NAT drugs vs combined drug loaded MSNs and combined drug loaded MSNs-si or ### p < 0.0001, combined drug MSNs vs combined drug loaded MSNs-si. Western blot analysis of mitochondrial protein responsible for inducing cell death. β -actin served as loading control. (e) Lane1- Control, Lane 2- NAT (ETO+CAR), Lane 3- MSNs (ETO+CAR), Lane 4- MSNs (ETO+CAR)-si. (f) Lane1- Control, Lane 2- NAT (DOC + CAR), Lane 3- MSNs (DOC + CAR), Lane 4- (DOC + CAR)-MSNs-si. 32


4

0.676

Page 33 of 36 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


b

a

** *** *** *** ***

c

1

3

2

d

4

Survivin

e

2

3

0.957

0.873

4

Survivin β actin

β actin Survivin/ β actin

1

1.01

0.841

0.606

Survivin/ β actin

0.51

NAT (ETO + CAR)

CONTROL 1.8

1.5

11.1

94.9

1.8

82.8

MSNs (ETO + CAR)

1.7

12.7

74.6

4.3

9.3

61.3

NAT (DOC + CAR)

1.1

0.6

8.9

96.6

1.7

80.5

MSNs (ETO + CAR-MSNs)-si

8.4

0.799

CONTROL

f 4.4

0.966

4.3

2.4

MSNs (DOC+CAR)-si

MSNs (DOC + CAR)

25.1

8.2

5.7

12.5

76.5

5.3

27.4

12.1

55.5

5.0

33


Cont.


g

Page 34 of 36

h

***

*** *** ***

***

***

Figure 5: qRT-PCR analysis of survivin gene following treatment. (a) NAT drugs (ETO + CAR), MSNs (ETO + CAR), MSNs (ETO + CAR)-si. (n=3: data as mean ± SD). ***p < 0.0001 control vs treatments. (b) NAT drugs (DOC + CAR) and MSNs (DOC + CAR), MSNs (DOC + CAR)-si. (n=3: data as mean ± S.D). **p < 0.008 or ***p < 0.0001, CONTROL vs Treatments. Western blot analysis of survivin protein. β -actin served as loading control. (c) Lane1- CONTROL, Lane 2- NAT (ETO + CAR), Lane 3- MSNs (ETO+CAR), Lane 4- MSNs (ETO+CAR)-si. (d) Lane1- CONTROL, Lane 2- NAT (DOC + CAR), Lane 3- MSNs (DOC + CAR), Lane 4- (DOC + CAR)-MSNs-si. Apoptosis assay in A549 cells by Annexin-V-PE and 7-AAD staining. (e) NAT drugs (ETO + CAR), MSNs (ETO + CAR) and MSNs (ETO + CAR)-si. (f) NAT drugs (DOC + CAR), MSNs (DOC + CAR) and MSNs (DOC + CAR)-si. Experiments were performed in triplicates and one of the representative picture has been provided. (g, h) Bar graph denoting the percentage of apoptosis following the above treatments. (n = 3: data as mean ± S.D). ***p < 0.0001, CONTROL vs Treatments.

34


Page 35 of 36 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


Table-1: IC50 (µg/ml) of single drug formulations in A549 cells NAT-ETO

35.7

ETO-MSNs

22.09

NAT-DOC

61.64

DOC-MSNs

51.78

NAT-CAR

5.11

CAR-MSNs

1.52

Table-2: IC50 (µg/ml) of combined drug formulations in A549 cells NAT (ETO + CAR)

0.514

NAT (ETO + CAR)-si

0.523

MSNs (ETO + CAR)

0.291

MSNs (ETO + CAR)-si

0.183

NAT (DOC + CAR)

6.95

NAT (DOC + CAR)-si

6.77

MSNs (DOC + CAR)

1.66

MSNs (DOC + CAR)-si

0.85

35



siRNA-drug loaded MSNs

Bcl 2

Mitochondria Bax

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

Page 36 of 36

Drug released and siRNA detached from MSNs

Nucleus Apoptosis Survivin protein synthesis blocked/suppressed


Augmented anticancer efficacy by si-RNA ... - ACS Publications

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