Telomerase Responsive Delivery of Doxorubicin from Mesoporous

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Telomerase Responsive Delivery of Doxorubicin from Mesoporous Silica Nanoparticles in Multiple Malignancies: Therapeutic Efficacies against Experimental Aggressive Murine Lymphoma Prateek Srivastava, Sumit Kumar Hira, Amod Sharma, Mohammad Kashif, Prashant Srivastava, Divesh N Narayan Srivastava, Ram Adhar Singh, and Partha Pratim Manna Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00342 • Publication Date (Web): 25 May 2018 Downloaded from http://pubs.acs.org on May 25, 2018

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Bioconjugate Chemistry

Telomerase Responsive Delivery of Doxorubicin from Mesoporous Silica Nanoparticles in Multiple Malignancies:

Therapeutic

Efficacies

against

Experimental Aggressive Murine Lymphoma Prateek Srivastava†‡⊥, Sumit Kumar Hira§⊥, Amod Sharma∥⊥, Mohammad Kashif†, Prashant Srivastava‡, Divesh Narayan Srivastava#, Ram Adhar Singh‡ and Partha Pratim Manna†* †

Immunobiology Laboratory, Department of Zoology, and ‡Department of Chemistry, Centre of

Advanced Study, Institute of Science, Banaras Hindu University, Varanasi 221005, India §

Department of Zoology, The University of Burdwan, Purba Bardhhaman-713104, India

∥Department

of Biological Sciences, Indian Institute of Science Education and Research Bhopal,

Bhopal-462 066, India #

CSIR-Central Salts and Marine Chemicals Research Institute, Bhavnagar 364002, Gujarat. India

*E-mail: [email protected]. KEYWORDS: Mesoporous silica nanoparticle, Telomerase, Cytotoxicity, Apoptosis, Therapy.

ACS Paragon Plus Environment

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ABSTRACT

Mammalian telomerase maintain the length and integrity of telomeres by adding the telomeric repeats to chromosome end. This work describes the telomerase responsive delivery of doxorubicin against telomerase positive human and murine cancer cells. Wrapping of doxorubicin loaded mesoporous silica nanoparticles with specific oligonucleotide sequence, containing telomeric repeat complementary sequence and a telomerase substrate primer sequence resulted slow and sustained release of doxorubicin, contiguous to the tumor cells. The DNA wrapped nanoprobe significantly inhibits the proliferation and enhanced the cytotoxicity in telomerase positive human and mouse tumor cells, and its function is impeded following exposure to specific telomerase inhibitor, AZT. Entrapping of doxorubicin by telomerase specific oligo, manifests enhanced apoptosis and significantly higher uptake of the drug in the tumor cells. Treatment of telomerase positive Dalton’s lymphoma bearing mice with a novel and newly designed oligo wrapped nano probe, specific for mouse telomerase, significantly enhanced the survival and improved the histopathological parameters. In addition, the treatment also induced significant reduction in the number of tumor foci and restored the normal architecture of the vascularised organs, besides preventing metastasis.


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INTRODUCTION Inorganic nanoparticles including mesoporous silica nanoparticles (MSNP) have been used for diverse biological purposes like drug 1, protein 2, gene delivery 3, cell imaging 4, diagnosis and bioanalysis 5. Due to large surface area, pore volume, stability, biocompatibility 6, high loading efficiency and ease of functionalization, the mesoporous silica serves as an excellent candidate for efficient nanocarrier. Various stimuli like pH 7, light 8, vitamins 9, redox potential 10and enzymes 11 were employed in regulating the efficient and controlled release of drug from MSNP. Among them, the enzyme mediated drug delivery system holds an advantage of higher levels of expression in the tumor cells, compared with the normal cells. Many drug delivery systems take benefit of certain enzymes like glutathione 12, esterase 13, MMP-9 14 and MMP-2 15 specific proteases, which showed promising results in cancer therapy and drug delivery related applications. Mammalian telomerase is another bullseye approach for exploring enzyme mediated drug delivery in telomerase positive cancers. Telomerase acts as a biomarker for cancer which functioned as reverse transcriptase, responsible for the addition of the polynucleotide "TTAGGG" to the 3' end of telomeres 16. Most of the somatic cells in human lack telomerase which resulted in shortening of the terminal repeat sequences, a cause for ageing 17. The telomerase repeat amplification protocol (TRAP assay) confirms over expression of telomerase which was observed in the nucleus as well as in the cytoplasm of neoplastic cells 18. This creates a possibility to develop telomerase specific delivery system against cancer. There are few reports which discussed the telomerase activity in releasing guest molecules from mesoporous silica nanoparticles 19. Recently, Y.Wan et al. 20 demonstrated the telomerase mediated glucose release from the specific DNA-gated mesoporous silica for bioanalysis assay. In an another study, the metal conjugated doxorubicin (DOX) loaded mesoporous silica was used as a traceable



nanocarrier which generated the surface enhanced Raman scattering (SERS) signals when drug release was mediated via telomerase responsive nanogated structure 21. Besides that, a series of methods were developed to detect the expression of telomerase in the nucleus as well as in the cytoplasm of neoplastic cells 22, 23. In our knowledge, the effect of telomerase activity as a drug releasing agent from MSNP in diverse, sensitive and drug resistant cancer cell lines has not been reported. Herein, we have synthesized MSNP with an average size of ~90 nm and are functionalized with the amine groups. Specific oligonucleotide sequences which contain telomeric repeat complementary sequence and a telomerase substrate primer sequence 19, seals the pores of DOX loaded aminated MSNP via electrostatic interactions. The nanocomposite of mesoporous silica and oligonucleotide provides excellent biocompatibility, and prevents premature drug release. The release of the oligonucleotides from the surface of the nanoparticle does not cause any harm to the cells. The wrapped oligonucleotides over the silica nanoparticles are specific to telomerase enzyme that elongates the oligonucleotide sequences. This resulted in the formation of rigid hairpin structure which subsequently leaves the surface of the nanoparticle and release DOX from the pores. The formed nano molecular probe (MSNP-NH2-DOX-DNA) significantly inhibited the proliferation of telomerase positive MCF-7 (human breast adenocarcinoma) 24, K562 (human erythroleukemia) 25 and DL (Dalton’s lymphoma) 26 cells. Telomerase negative U2OS (human osteosarcoma) 27 cells remain unresponsive to the construct, likely due to the absence of the enzyme. In U2OS cells, free DOX treatment demonstrated markedly higher antitumor effect compared with the nano probe. Treatment with telomerase inhibitor AZT 28 obliterated the role of telomerase in the nano probe, indicating active participation of the enzyme. Oligonucleotide wrapped nano probe augmented the uptake of doxorubicin in DL, K-


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562 and MCF-7 cells along with the significant apoptosis compared with equimolar treatment with free doxorubicin. For human tumor cells (MCF-7, K-562, and U2OS) and normal human lymphocytes, monocytes and RBC, we used nano probe constructed with human specific DNA oligo, wrapped on MSNP-NH2-DOX. We have designed and synthesized a novel mouse specific DNA oligo for study against DL and DLR cells. Therapy with DNA wrapped nano probe significantly augmented the survival of tumor bearing animals and reduced the tumor mass with no significant increase in abdominal circumference and body weight. Besides that, treatment with the DNA wrapped molecular nano probe reduced the tumor burden in vascularised organs like liver, spleen and lung and improved the histopathological parameters. RESULT AND DISCUSSION Human cancer cells own active telomerase and prevent shortening of telomeres during proliferation. The requirement for active telomerase duty is essential due to the occurrence of chromosome aneuploidy and dynamic replicative cycles. Most of the somatic cell lines lacks active telomease or have telomerase insuffciency. TRAP assay/PCR confirms the high concentration of telomerase in cytoplasm and nucleus of cancer cells. As mentioned earlier, telomerase can be used as an modulator in releasing guest molecules, thus the effect of telomrease activity in releasing the drug (DOX) from the nanoparticles against cancer cell lines can be advantageous. Among the various nanoparticles, mesoporous silica nanoparticles are choosen as nanomedicine model due to its stability, ease of functinalization, high drug loading efficiency and excellent biocompatibility. For attaining the telomerase activity, specific oligonucleotide sequence [5ˊ-(CCCTAA)n AATCCGTCGAGC AGAGTT-3ˊ] which consist of complementary repeat sequences and the primer sequences are grafted over the amine functionalized MSNP. Telomerase acts on the nanoparticles and with the aid of dNTPs, it tends



to elongate the oligonucleotide sequence in 5ˊ- 3ˊ direction. The added repeat DNA sequence TTAGGG at the 3ˊ end hybridised with the complementary CCCTAA sequence at the 5ˊ end and formed a rigid, hairpin-like DNA structure which leaves the surface of the nanoparticles, causing drug release through the pores. We were interested to investigate the release of the guest molecule from the silica nano device employing the telomerase specific DNA oligonucleotide. The specific oligo is comprised of two parts. To understand the efficacy of the construct in liberating the DOX molecules inside the cancer cells, we investigated the role of the construct for in-vitro and in-vivo (DL tumor bearing AKR/J mouse) tumoricidal applications. Herein, both human and mouse specific DNA oligos were designed, which have the specificity towards the telomerase enzyme. Since the mouse telomerase pertains to different template RNA sequence as compared to human, thus we designed a novel mouse specific DNA oligo as represented in the Scheme 1. We further generated the secondary structures of the mouse and the human specific DNA oligo, using the mfold web server at default settings (Figure S1 & S2). We also predicted the hairpin loop structures by the software for both human and mouse constructs, subsequent to the addition of the telomeric repeats to the DNA oligo primers via telomerase enzyme.This results in unwrapping of MSNP and release of the drug (Figure S3 & S4).


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Scheme1. Schematic illustration of MSNP nanoprobe construct using oligonucleotide sequence containing telomeric repeat sequence for intracellular analysis of telomerase. Red color indicate the stretch of four nucleotides template region of telomerase of human or mouse at 3’ end. MSNP was synthesised via sol-gel chemistry. The as synthesised silica nanoparticles showed average size of ~90 nm,demonstrated by transmission electron microscopy (TEM) image presented in Figure S5A. The Figure S5B represents the lattice fringes in the silica nanoparticles for the cargo loading. The nitrogen adsorption desorption isotherm of the MSNP revealed a typical type IV isotherm representing porous characters with specific surface area of 1243 m2/gm and BJH pore size of 3.3 nm (Figure 1A). The amine functionalized MSNP (MSNP-NH2) is important for various biological attachment. The DOX loading efficiency and its loading content in the construct (MSNP-NH2-DOX) were 66% and 5.9% respectively. In order to prevent the premature drug release, it is important that effective wrapping with DNA is made for sealing the pores of MSNP-NH2. The wrapping of DNA over the amine terminated



MSNP via electrostatic interactions was evaluated using UV-vis spectroscopy, Zeta potential measurements and Fourier-transform infrared spectroscopy (FT-IR) study. The DNA showed absorbance band in UV range (240-260 nm) which was evident form the peak at 256 nm, when the DNA was attached to MSNP (MSNP-NH2-DOX-DNA) (Figure 1B). Free doxorubicin shows two strong UV absorption maxima at 233 nm and 252 nm as represented in Figure 1B. However, when DOX molecules were caged inside the MSNP, a shift in the UV absorption was manifested approximately at 218 nm in MSNP-NH2-DOX. The amine terminated silica formed strong molecular interactions with the negatively charged oligo biomolecules which adsorbed over the surface through the non-covalent forces. The de novo synthesised oligo wrapped silica displayed a strong UV absorption band at 253 nm on account of the purine and pyrimidine rings in the oligo structure. The peak at 1563 cm-1 in the Infrared spectra (IR) represents N-H bending vibration which confirmed the attachment of the amino propyl group over the MSNP (Figure1C). Further, the positive Ninhydrin staining of the amine functionalized MSNP was in agreement with the IR data. The oligonuclotides gave specific peaks at 1180 cm-1 and 1636 cm-1 which corresponds to the asymmetrical streaching vibrations of PO2- and C=O/C-N and bending vibration of N-H groups in the IR spectra respectively. The oligonucleotide wrapping over the surface of MSNP caused the disapperance of amine group peak at 1563 cm-1 in the IR spectra (Figure 1C), suggesting the possible molecular interactions between the amine groups of MSNP and the phosphate groups present on the oligonucleotide.


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Figure 1. Nitrogen Adsorption-Desorption isotherm of MSNP and distribution of BJH pore size (A). UV absorption spectra recorded in free doxorubicin, MSNP-NH2, MSNP-NH2-DOX and MSNP-NH2-DOX-DNA (B). FT-IR Spectra of bare MSNP, MSNP-NH2, free oligo and MSNPNH2-DNA (C). Number of experiments performed (n) = 3. Dynamic light scattering (DLS) represents the hydrodymaic size of the nanoparticles in the aqueous solution. The DLS analysis for the MSNP-NH2 and the oligo-wrapped MSNP showed average size of ~122 nm and ~163 nm respectively as determined in PBS (10 mM, pH 7.2) (Figure S6A). The augmentation in size of the particles can be ascribed to the wrapping by the telomerase specific oligonucleotides over the external surface of MSNP which increased its



hydrodynamic size. The study of Zeta potential is an effective way to confirm the sucessful functionalizations over the surface of nanoparticles as it changes during every step of fabrication. The zeta potential was conducted in PBS (10 mM) solution at the physiological pH 7.2. The zeta potential for bare MSNP was -27.8±0.87 mV due to the presence of hydroxyl groups. When the amine groups were attached over the MSNP surface, the zeta potential become positive to +8.1±0.310 mV (MSNP-NH2). Following the wrapping of oligonucleotide over the amine terminated MSNP, the Zeta potential again switched to negative -25±1.012 mV (MSNP-NH2DNA) (Figure S6B). We have tested the effects of the oligonucleotide wrapped nano probes (human and mouse) against telomerase positive human and murine tumor cells to assess their role with respect to loss of viability and growth inhibition. Telomerase positive DL, MCF-7, and K-562 cell’s viability was declined significantly following treatment with oligoneucleotide wrapped nano probe (MSNP-NH2-DOX-DNA) compared with free doxorubicin. (Figure 2, Figure S7 & S8). DL cells treated with the mouse specific probe (50 µM) reduced the viabiity to 60% compared to 20% following treatment with free DOX (p

Telomerase Responsive Delivery of Doxorubicin from Mesoporous

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