Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells

    Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells Mehul Jivrajani, Manish Nivsarkar PII: DOI: Reference:

S1549-9634(16)30077-6 doi: 10.1016/j.nano.2016.06.004 NANO 1361

To appear in:

Nanomedicine: Nanotechnology, Biology, and Medicine

Received date: Revised date: Accepted date:

31 January 2016 13 May 2016 14 June 2016

Please cite this article as: Jivrajani Mehul, Nivsarkar Manish, Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the efficient and cost effective delivery of shRNA to cancer cells, Nanomedicine: Nanotechnology, Biology, and Medicine (2016), doi: 10.1016/j.nano.2016.06.004

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ACCEPTED MANUSCRIPT Revised manuscript Ligand-targeted bacterial minicells: Futuristic nano-sized drug delivery system for the

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efficient and cost effective delivery of shRNA to cancer cells

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Mehul Jivrajani1, 2 and Manish Nivsarkar1, *

Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and

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Research Development (PERD) Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad, 380 054, Gujarat, India.

Faculty of Science, NIRMA University, Sarkhej-Gandhinagar Highway, Gota, Ahmedabad-

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382 481, Gujarat, India.

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Corresponding author

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Manish Nivsarkar, Ph.D.

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E-mail address: Mehul Jivrajani- [email protected]

Director,

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B.V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Sarkhej-Gandhinagar Highway, Thaltej, Ahmedabad-380 054, Gujarat, India. Tel: +917927416409; fax: +917927450449,

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E- mail address: [email protected]

Word Counts: Abstract Word Count = 146; Manuscript Word Count = 4998; Number of References = 38; Number of Figures / Tables = 8/1

Acknowledgment The authors are thankful to B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, Ahmedabad for providing all the facility for the successful completion of the work. Authors are also grateful to Dr. Lawrence Rothfield (University of Connecticut, USA) for providing E.coli PB114 as a generous gift. Additionally, authors would like to thank CSIR, India for providing financial assistance to Mehul Jivrajani as Senior Research Fellowship (# 113353/2K12/1) to carry out this work. Authors are also thankful to NIRMA University, Ahmedabad, Gujarat, India. 1

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The authors have no conflict of interest and no disclosures.

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Presentation of abstract at meeting:

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Jivrajani M et al. Minicell packaged targeted delivery of shRNA to cancer cells. European journal of cancer, 2014 50(6); 85-86

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26th EORTC-NCI-AACR Symposium on Molecular Targetds and Cancer Therapeutics

Abstract:

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In this study, shRNA against VEGFA was packaged in bacterial minicells and surface of minicells was modified with folic acid. Analysis of cellular internalization revealed that folic acid conjugated minicells internalized through receptor mediated endocytosis in folate and PSMA receptor positive KB and LNCaP cells, respectively. In contrast, A549 (folate receptor negative) cells showed minute internalization. In vitro delivery of

FA

minicellsVEGFA

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significantly reduced the expression of VEGFA mRNA in KB and LNCaP cells whereas expression of VEGFA remained unaltered in A549 cells.

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minicellsVEGFA significantly

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reduced tumor volume in mice bearing KB and LNCaP xenograft. On contrary, gradual increase in the tumor volume was recorded in A549 xenograft. FAminicellsVEGFA significantly

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silenced the VEGFA mRNA in KB and LNCaP xenograft. Expression of VEGFA remained same in FAminicellsVEGFA delivered A549 xenograft. In vivo biodistribution study showed that FA

minicellsVEGFA were localized in the tumor followed by intravenous

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majority of

administration.

Graphical abstract 

Schematic representation of VEGFA specific shRNA packaging into minicells, folic acid conjugation on minicell surface and its in vitro and in vivo delivery.



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minicellsVEGFA were effectively taken up by receptor mediated endocytosis and

silenced expression of VEGFA in folate receptor positive cell lines. 

In vivo delivery of

FA

minicellsVEGFA significantly reduced tumor volume which could

be attributed to reduced angiogenesis followed by downregulation of VEGFA. 

In vivo biodistribution demonstrated that majority of to tumor followed by passive and active targeting. 2

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minicellsVEGFA were targeted

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Folic acid conjugated minicells offers a great potential for in vivo targeted delivery of

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shRNA to cancer cells.

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Keywords: Bacterial minicells; shRNA delivery; folate receptor targeted delivery; RNA

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interference; cancer

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ACCEPTED MANUSCRIPT Background RNA interference (RNAi) became the most fascinating tool for the researchers to develop newer targeted therapy for the various diseases, after it’s revolutionary discovery by Fire and

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Mello in 1998 [1]. Fire and Mello showed that dsRNA could specifically silence targeted

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endogenous mRNA before translation and induce genetic interference [1]. This landmark discovery propelled RNAi from an ‘interesting’ cellular phenomenon to a technology so powerful that it formed a totally new class of treatment for various diseases. RNAi has

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emerged as a novel therapy in many diseases where overexpression and/or faulty expression of genes are responsible for the occurrence of disease. Cancer is an example of disease where

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many genes are overexpressed or mutated. Downregulation of such genes by RNAi restrain tumor cell proliferation, metastasis and eventually reduce the tumor growth. RNAi can be induced artificially by introducing synthetic si(short interfering) RNAs or sh(short hairpin) RNAs into mammalian cells [2]. Hence, RNAi has potential to silence virtually any disease

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causing gene in the cells.

In spite of significant potential of si/shRNA therapy, most approaches to RNAi-mediated

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gene silencing for cancer therapy have been with cell cultures in the laboratory, and key impediment in the transition to the clinic due to lack of appropriate delivery system still need

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to be addressed [3]. The main challenges that need to be overcome for exogenous RNAi to be widely used as a cancer therapeutic include need for efficient design of active si/shRNAs to ensure optimum gene silencing activity with negligible off-target effects, poor stability due to

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rapid degradation by serum nucleases, non-specific tissue distribution, poor membrane permeability of si/shRNAs limiting it’s cellular uptake for in vivo targeting [4–6].

To bestow with drug-like properties such as stability, tissue bioavailability and cellular delivery to si/shRNA, several strategies have been developed such as viral vectors [7], aptamers [8], lipid based delivery [9], and polymer mediated delivery [10]. However, all of these delivery systems have one or more problems such as degradation product of delivery systems is unsafe, immune toxicity, reduced penetrating efficacy, poor stability, etc., which has minimized their use [4,11].

MacDiarmid et al. described a new approach i.e. use of bacterial minicells for encapsulation and cancer cell targeting of si/shRNA. [12]. Minicells have several advantages over other 4

ACCEPTED MANUSCRIPT delivery systems such as wide variety of chemotherapeutic drug with variable physicochemical and structural characteristics can be efficiently packaged, convenient drugloading process, loading of sufficient amount of siRNA (~12000) and shRNA (~100 copies),

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easy surface with antibodies/ligand for active targeting, etc [13]. si/shRNA packaged

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minicells can be targeted to tumor cell-surface receptors by bispecific antibodies linked on the minicells. Receptor engagement results in minicell endocytosis, intracellular degradation, and si/shRNA release. Consequently, significant tumor growth inhibition and regression was

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achieved through the suppression of targeted gene [12]. Recently, EGFR targeted minicells (EDV™ nanocells) have also been exploited for the delivery of microRNA (miR-7 and miR-

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193a-3p) [14,15]. Eventhough this approach looks promising, use of bispecific antibodies for the active targeting makes it more complex and expensive. Generation of bispecific antibodies itself is a costly affair and require substantial expertise for it’s characterization and selection. Further, use of mouse chimeric antibodies may elicit immune response [16]. Moreover, polymorphisms and genetic mutation in the targeted receptor may alter the

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specific binding and subsequent internalization of cargo linked with antibodies. For instance, mutant variant of EGFR receptor, EGFRvIII is accountable for impaired internalization and

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sorting to lysosomes which contribute to resistance against EGFR receptor targeting and treatment failure [17, 18]. Additionally, cetuximab is only efficacious to wild-type EGFR

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receptor and lack efficacy in patient bearing KRAS mutation in colorectal cancer [19]. Similarly, Di Nicolantonio et al. showed that BRAF mutations (BRAF V600E allele) is responsible for the failure of response to either cetuximab or panitumumab [20]. In view of

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these challenges, versatile yet efficient and cost effective targeting approach is required to develop minicells mediated targeted drug delivery system more adaptable. Hence, this problem can be overcome by the use of targeting ligand which is essential for the survival and/or proliferation of cancer cells, for instance folic acid; for the active targeting of minicells.

In this study, for the first time to the best of our knowledge, an attempt was made to develop folate receptor targeted minicells for the shRNA delivery in cancer. Here, tumor angiogenesis was targeted through the folate receptor targeted minicells packaged with shRNA specific for vascular endothelial growth factor A (VEGFA). Schematic representation of shRNA packaging into minicells, folic acid conjugation and subsequent analysis is depicted in the supplementary fig. 1 (Graphical abstract). Initially, in vitro delivery and uptake mechanism of 5

ACCEPTED MANUSCRIPT this delivery system was investigated. Subsequently, efficacy of this delivery system was evaluated in vitro in several human cancer cell lines and in vivo in mice tumor xenograft model. Eventually, in vivo biodistribution of folate receptor targeted minicells was evaluated

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in mice bearing tumor xenograft.

Methods Materials

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All the reagents and kits used in this work provided in the supplementary materials.

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Cell lines

Cancer cell lines, A549, KB, and LNCaP have been procured from National Centre for Cell Sciences (NCCS), pune, India. KB and LNCaP have been selected as positive control due to high level of folate receptor expression whereas A549 was selected negative control, which has very least amount of folate receptor expression [21]. Culture conditions for all the cell

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lines are mentioned in the supplementary material.

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Animals

Healthy mice C57 BL/6 were purchased from Mahaveera Enterprises, Hyderabad, India. All

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the mice were kept in individually ventilated cages (IVC), with a relative humidity of 60 ± 5% and a temperature of 25 ± 2˚C. A 12:12 h light : dark cycle was also regulated for these

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animals. Autoclaved balanced rodent food pellet and water was provided ad libitum. All experimental protocols were reviewed and accepted (PERD/IAEC/2013/014) by the Institutional Animal Ethics Committee prior to initiation of the experiment.

Expression vectors Plasmid based expression vectors such as pSUPERneo, pRNAT and vector having VEGFA shRNA were kindly provided by Dr. Neeta Shrivastava (Department of Pharmacognosy and Phytochemistry, PERD centre, Ahmedabad, Gujarat, India). pSUPERneo is a backbone vector in which shRNA sequence specific for VEGFA and scramble sequence (control shRNA) have been cloned. siRNA sequence for VEGFA and scramble was 5’-AAT CAT CAC GAA GTG GTG AAG-3’ and 5’- AAC AGT CGC GTT TGC GAC TGG-3’, respectively. pRNAT is an expression vector having GFP (Green Fluorescent Protein) as a reporter gene under the U6 promoter. 6

ACCEPTED MANUSCRIPT Characterization of minicells producing bacterial strain E.coli PB114, minicell producing mutant strain was kindly provided by Dr. Lawrence Rothfield, University of Connecticut, USA. Enrichment of E.coli PB114 was carried out in

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LB broth supplemented with 50 µg/ml kanamycin. Gram’s staining was performed in order to

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observe the morphology and presence of minicells. Subsequently, cells were further characterized by Transmission Electron Microscopy (TEM) using Transmission Electron

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Microscope (Tecnai 20, 200 kv, Philips, Holland) at an accelerating voltage of 90 kV.

Packaging of shRNA expression vector and minicell purification

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shRNA expression vectors, namely pSUPERneoScramble (designated as scramble) and vector having shRNA specific for VEGFA (designated as VEGFA) have been transformed by calcium chloride method into the E.coli PB114 in order to package these vectors into the minicells.

Subsequently, minicells packaged with shRNA vectors were purified from it’s respective

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parent bacterial cells by previously reported method by us [22]. Purity of minicells was confirmed by platting final fraction on LB agar plate. Eventually, purified minicells were

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grown for 14 days in thioglycolate broth to confirm the absence of any slow-growing organisms in the final fraction. Purified minicells were stained with FITC and characterized

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by fluorescence microscopy and TEM. Further, packaging of shRNA expression vector was confirmed by isolation of plasmid DNA from the purified minicells by alkaline lysis method

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and subsequent gel electrophoresis and spectrophotometric analysis.

Folic acid conjugation Folic acid conjugation reaction was performed with several modification of previously reported method [23]. Detailed method has been mentioned in the supplementary material. Folic acid conjugated minicells were stained with FITC and observed in fluorescent microscope (Olympus BX 41, Osaka, Japan) and TEM for its integrity. Subsequently, average size of folic acid conjugated minicells was determined by dynamic light scattering method (Zeta sizer ZS 90, Malvern).

In vitro delivery of folic acid conjugated minicells Detailed method has been mentioned in the supplementary material.

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ACCEPTED MANUSCRIPT Mechanism of minicell endocytosis Cells (KB and LNCaP) were grown in folate free medium at a density of 106 cells per plate in a 24 well plate and treated with 109 FITC loaded minicells as respective treatment 1) FA

minicellsVEGFA at 37°C, 3)

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minicellsVEGFA at 4°C and 4)

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minicellsVEGFA at 37°C, 2) FA

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minicellsVEGFA with free folic acid at 37°C for 1 hour in humidified incubator with 5%

carbon dioxide and 95% air. After incubation, all cells have been washed thrice with PBS and observed under fluorescence microscope. Moreover, uptake of minicells was also evaluated

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quantitatively (in terms of FITC) by spectrofluorometer (SL174, Systronics India Ltd.). Cells were treated with 1% Triton X 100 for 20 min at 4°C to release FITC. Total protein

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concentration of cells was determined by BCA method. Cell lysate was analysed by spectrofluorometer at excitation and emission wavelengths of 490 and 515, respectively. Total fluorescence (mV) was represented per mg of protein.

Subsequently, uptake of

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minicellsVEGFA was also analyzed in the presence of several

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cellular uptake inhibitors to reveal the mechanism of endocytosis. Cells were seeded as described above. Cells were pre-treated with different uptake inhibitors such as sodium azide

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(0.1%), colchicine (2 µg/mL), filipin complex (4 µg/mL), and phenylarsine oxide (0.5 µg/mL), into each group of wells separately and incubated at 37°C for 10 min. Then the FA

minicellsVEGFA were delivered in the presence

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medium was removed and 109 FITC loaded

of different endocytosis inhibitor (at same concentration ) into the corresponding group of

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wells and incubated at 37°C for 30 min in humidified incubator. After incubation, the medium was removed and cells were washed three times with PBS and observed under the fluorescent microscope. Eventually, minicells endocytosis was also determined quantitatively by spectrofluorometer as described above.

In vitro delivery and qualitative gene expression analysis Cells (A549, KB and LNCaP) were grown in folate free medium as described above, a day before study. Next day all the cells have been treated with 109 FAminicellspRNAT and incubated in humidified incubator for 1 hour. After incubation, all cells were washed three times with PBS to remove free

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minicellspRNAT and replenished with respective growth medium. Cells

were observed under fluorescence microscope for the expression of GFP after 72 hours.

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ACCEPTED MANUSCRIPT In vitro delivery of FAminicellsVEGFA and gene expression Cells were grown in folate free medium as described above, treated with FA

minicellsscramble 2) minicellsVEGFA, 3)

109 1)

FA

minicellsVEGFA and incubated for 1 hour in

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humidified incubator with 5% carbon dioxide and 95% air. After incubation, cells were

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washed thrice with PBS and replenished with respective medium. After 72 hrs, cells were processed for the gene expression analysis of VEGFA by PCR.

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In vitro gene expression by PCR

VEGFA gene expression levels were determined by semi-quantitative reverse-transcription

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PCR. Total RNA was isolated from the different groups of A549, KB and LNCaP cells using TRIzol reagent. RNA samples were quantified by OD 260/280 and 1 µg RNA was used to synthesized cDNA. cDNA was amplified using primer specific for VEGFA, 18s and GAPDH (Table. 1). Amplified PCR products were analysed on 1.5% agarose gel, relative band intensities of various PCR products were calculated using Quantity One 4.6.9 (Basic)

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software (Bio-Rad, Hercules, California, USA) and represented as relative gene expression.

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(Detailed method has been mentioned in the supplementary material).

Development of tumor xenograft in mice Tumor xenograft was developed in immunocompromised C57BL/6 mice as described in the

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recently reported protocol by us [24]. Briefly, 0.1 ml of cells (approximately 5×106 A549, KB and LNCaP cells) were subcutaneously injected into the shaved right shoulder blade of

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mice. Tumor volume was measured every week externally by digital caliper using following formula [25]:

Volume (mm3) = (A) × (B2)/2, where A was the largest diameter (mm) and B the smallest (mm) In vivo delivery of FAminicellsVEGFA and gene expression All the mice bearing tumor xenograft of A549, KB and LNCaP have been randomized on the basis of tumor volume (80 to 100 mm3) and divided into four groups (n=6). Animals were divided in the following groups such as 1) Saline, 2)

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minicellsscramble, 3) minicellsVEGFA, 4)

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minicellsVEGFA. 109 of respective minicells have been administered intravenously through

tail vein on 1st and 3rd week. Tumor volume was measured every week externally by digital

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ACCEPTED MANUSCRIPT caliper as described above. At the end of treatment tumor regression was recorded, tumors were excised and relative angiogenesis was also recorded in all the group of animals.

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Eventually, tumors were excised from each group of animals, 50 mg of tumor tissue was

homogenized

with

rotor-stator

homogenizer

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weighed and snap frozen in liquid nitrogen. 1 ml of TriZol reagent was added and tissue was (polytron,

kinematica,

luzern

Switzerland) at 10000 rpm (2 cycles for 30 sec each). Remaining procedure was same as

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described above.

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Analysis of cytokine and interferon response

Detailed method has been mentioned in the supplementary material. In vivo biodistribution of FAminicellsVEGFA Biodistribution of

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minicellsVEGFA was investigated in mice bearing KB tumor xenograft. FA

minicellsVEGFA intravenously through tail

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Mice were administered with 109 FITC loaded

vein (n=6). 3 hrs post injection, animals were sacrificed by CO2 asphyxation. Vital organs

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such as liver, heart, lung, kidney, brain, spleen along with tumor were excised and snap frozen in liquid nitrogen. Subsequently, cryosection were taken using Cryotome Cryostat

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(Leica, CM 1900, Wetzlar, Germany). Sections from same tissue were kept unstained for the fluorescence microscopy and stained with H & E for the light microscopy.

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Statistical analysis

All the data were represented as mean ± SD. One-way ANOVA followed by Bonferroni correction posthoc test was applied to determine the significant difference among groups. Probability values with p ≤ 0.05 were considered to be significant.

Results Characterization of minicells producing bacterial strain, shRNA packaging, minicells purification and folic acid conjugation

It can be observed from the fig.1(a) that E.coli PB114 turned pink upon staining thus confirming the presence of gram negative organism. It also showed the presence of large number of minicells which are indicated with arrows. Further, detailed morphology and 10

ACCEPTED MANUSCRIPT minicells production was observed more closely by TEM. Fig. 1(b) depicts sequential event of minicell production from parent cell which is indicated by arrows. Fig.1(b)(i) shows bacterium in which minicell production was just initiated from the pole. Fig. 1(b)(ii) shows

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minicell in the stage of its division from parent bacterium. Fig.1(b)(iii) shows minicell

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divided from the parent bacterium. Fluorescence microscopy images of minicells revealed large number of purified minicells present per field (Fig. 1(c)). TEM image showed that

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minicells had intact structure and of well-formed spherical shape (Fig.1(d)).

Fig. 1(e) shows the gel electrophoresis of isolated plasmid DNA along with purified plasmid

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DNA. Both the isolated plasmid shows corresponding bands with purified standard scramble and VEGFA plasmid DNA which confirmed that both the plasmid expression vectors have been segregated into the minicells.

Fig. 1(f) shows schematic representation of folic acid conjugation on minicells. Successful

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conjugation of folic acid was qualitatively confirmed by the permanent change in color of minicell pellet. Fig. 1(g) shows images of minicell pellets, before and after folic acid

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conjugation reaction. Folic acid imparted its yellow color on minicells after the successful conjugation. It can be observed from the image that minicell pellet turned yellow in color

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after the folic acid conjugation. Moreover this yellow color did not fade out even after repeated washing with PBS. This confirmed the folic acid conjugation on the minicells. Further, amount of conjugated folic acid was found to be ~ 2.0 ± 0.112 mg/1010 minicells.

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TEM image showed that minicells were morphologically similar with unconjugated minicells with well characterized cell wall and cell membrane (Fig. 1(h)). Average size of folic acid conjugated minicells was found to be 543.2 nm (Fig.1 (i)).

In vitro delivery of folic acid conjugated minicells Fig. 2 shows that, there was a very minute amount of minicells uptake as evident by very weak fluorescence intensity in case of A549 cells. In contrast, KB cells showed large amount of minicells uptake as demonstrated by intense fluorescence. Similarly, LNCaP cells also demonstrated considerable amount of minicells uptake. Additionally, in both the cell lines (KB and LNCaP), uptake of minicells was concentration dependent. Further, fluorescence and phase contrast overlaid image of KB cells after delivery of

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FA

minicellsVEGFA revealed the

ACCEPTED MANUSCRIPT large number of minicells uptake which confirmed the in vitro delivery of

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minicellsVEGFA

(Supplementary Fig.3).

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Mechanism of minicell endocytosis

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It can be visualized from the fig. 3 (a, b) that in both the cell lines, there was a large amount of minicells uptake as evident by increased fluorescence intensity when incubated at 37°C. In contrast, there was seldom uptake of minicells in any of the cell lines when incubated at 4°C.

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Similarly, when minicells were delivered without targeting moiety, i.e. folic acid, there was no uptake in any of the cell lines. However, delivery of folic acid conjugated minicells in

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presence of free folic resulted in the decreased fluorescence intensity and hence significant reduction in the uptake of minicells. These results were further confirmed when cell lysate was analysed quantitatively to measure extent of uptake in terms of total fluorescence. As shown in fig.3 (c, d), a clear differential uptake between folate targeted and non-targeted minicells was observed. At low temperature (4°C), there was extremely least uptake of FA

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minicellsVEGFA. Similarly, addition of free folic acid into the medium reduced the uptake of

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minicellsVEGFA.

Fig. 3 (e, f) shows that treatment with sodium azide and phenylarsine oxide inhibited the FA

minicellsVEGFA whereas minicells were readily taken up by the cells treated with

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uptake of

filipin and colchicin. Mean fluorescence shows that endocytosis of FITC loaded FA

minicellsVEGFA in presence of sodium azide and phenylarsine oxide was considerably lower

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than endocytosis in presence of filipin, colchicin and untreated control in both the cell lines (Fig. 3 (g, h). Moreover, reduced fluorescence in cells treated with sodium azide and phenylarsine oxide were statistically significant as compared to untreated control.

In vitro delivery and qualitative gene expression analysis It can be observed from the fig. 4 (i) that A549 cells have hardly one or two GFP positive cells per field after delivery of

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minicellspRNAT. In contrast, cell lines such as KB and

LNCaP have large number of GFP positive cells present per field (fig. 4 (ii and iii). In vitro delivery of FAminicellsVEGFA and gene expression Fig. 5(a-c) shows results of gene expression analysis by PCR after in vitro delivery of 1) FA

minicellsscramble, 2) minicellsVEGFA, 3)

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minicellsVEGFA. It can be visualized from fig. 5(a) 12

ACCEPTED MANUSCRIPT that expression of VEGFA did not change in any of the group in A549 cells. Contrary, expression of VEGFA reduced significantly in when compared with

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minicellsVEGFA treated KB and LNCaP cells

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minicellsscramble, and minicellsVEGFA treated cells (fig. 5 (b, c).

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Moreover, delivery of scramble shRNA did not change the expression of VEGFA in any of

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the selected cell lines. In vivo delivery of FAminicellsVEGFA

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Fig. 6(a, b and c) shows photographs of mice bearing tumor xenograft from each group at start (A) and end (B) of the treatment from A549, KB and LNCaP xenograft, respectively.

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Similarly, fig.6 (d, e, and f) shows graphs of mean tumor volume at every week during the treatment from A549, KB and LNCaP xenograft, respectively. It can be observed from the fig. 6 (a) and (d) that in case of A549 xenograft there was a gradual increase in the tumor volume till the end of treatment in all four groups. Whereas in case of KB and LNCaP xenograft, there was a significant decrease in tumor volume in FAminicellsVEGFA treated group

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as compared to other groups (fig. 6 (b, c, e, f)). Moreover, these differences in tumor volume were statistically significant. These results were further confirmed when tumors were excised

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from each group of animals to observe the relative angiogenesis (Supplementary fig. 5).

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In vivo gene expression by PCR

Fig. 7 (a-c) shows results of gene expression analysis by PCR after in vivo delivery of 1) saline, 2)

FA

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minicellsscramble, 3) minicellsVEGFA, 4)

minicellsVEGFA. It can be visualized from

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fig. 7 (a) that expression of VEGFA did not change after the delivery of

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minicellsVEGFA in

A549 xenograft which is in harmony with in vitro delivery studies. In contrast, expression of VEGFA decreased significantly in

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minicellsVEGFA treated group when compared with other

groups in KB and LNCaP xenograft (fig.7 b and c). Moreover, these results were in synchronization with tumor regression and reduced angiogenesis in KB and LNCaP xenograft. Additionally, in

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minicellsVEGFA treated

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minicellsscramble and minicellsVEGFA treated

groups, expression of VEGFA were comparable with saline treated group in all the three xenograft.

Analysis of cytokine and interferon response As shown in the supplementary fig.6, levels of IFN-γ, TNF-α and IL-6 were found to be significantly higher as compared to saline treated animals at 3 hr. However, at 24 hr, only 13

ACCEPTED MANUSCRIPT TNF-α level was found to elevated whereas levels of IFN-γ and IL-6 returned to normal range.

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In vivo biodistribution of FAminicellsVEGFA organs 3 hr post intravenous delivery of

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Fig. 8 shows fluorescence and corresponding H & E stained images of tumor and all vital FA

minicellsVEGFA. It can be examined from the

fluorescence image that majority of fluorescence was localized in the tumor. Cryosection of

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liver and heart also showed considerable fluorescence, however it was majorly restricted in blood vessels (indicated with arrow). Cryosection of spleen also showed very weak

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fluorescence. Contrary, cryosection of kidney, brain, lung and skin did not show any fluorescence.

Discussion

In this study, we have developed folic acid conjugated minicells for the in vivo targeted

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delivery of shRNA to cancer cells. Folic acid is a vitamin B9 essential for the de novo nucleotide synthesis. Therefore, the ability to acquire folate is imperative for the survival of

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rapidly dividing cancerous cells [26]. The cellular transport of folate is mediated by the folate receptors. Folate receptors have been found to be overexpress on cancer cell surface in

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majority of malignancies such as ovary, uterus, breast, kidney, bladder, brain, head and neck carcinomas and myeloid leukaemia [21, 27]. The receptor is generally absent in most normal tissues with the exceptions of choroids plexus in brain, placenta, and low levels in lung,

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thyroid, and kidney [26]. Further, folic acid is inexpensive, easy to conjugate with variety of biomaterials and non-immunogenic in humans [28]. These desirable features of folic acid make it as one of the most preferred choice of ligand for the development of targeted drug delivery systems [28–31].

Here, we have targeted angiogenesis through the silencing of pro-angiogenic factor, VEGFA. Angiogenesis is critical for the growth and metastases of tumors. Among various proangiogenic factors, VEGFA is the predominant pro-angiogenic factor [32]. Inhibition of VEGFA reduces the formation of new blood vessels, cuts off the supply of oxygen and nutrients to the tumor which lead to tumor stabilization or regression. Hence, silencing of VEGF A through RNAi offers a promising and novel class of therapeutics for various kinds of solid tumors. 14

ACCEPTED MANUSCRIPT Gel electrophoresis of isolated plasmid DNA from minicells confirmed the segregation and packaging of shRNA expression vectors into the minicells (fig.1 e) and yielded ~145 ± 6 and ~113 ± 8 plasmid copies for scramble and VEGFA plasmid DNA per minicell, respectively;

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which is in harmony with the previous report [12]. In the next step, folic acid was conjugated

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with bacterial minicells’ surface protein for active targeting to folate receptor. To evade possible damage to the minicells by the harsh coupling agent (DCC and NHS), folic acid conjugation was performed in aqueous medium using mild water soluble EDC and sulfo-

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NHS coupling agents. EDC reacted with γ carboxyl group of folic acid to form O-acylisourea intermediate which reacted with free amine present on the minicell surface to form amide

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bond. However, O-acylisourea intermediate is highly unstable and quickly hydrolysed to form isourea. But presence of sulfo-NHS in the reaction reacted with γ carboxyl group of folic acid to produce amine reactive sulfo-NHS ester. This in turn reacted with free amine present on the minicells surface proteins to form covalent amide bond and produced folic acid conjugated minicells. Addition of DMAP in the reaction increased the yield of folic acid

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conjugated minicells.

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In vitro delivery of folic acid modified minicells showed the uptake in folate receptor positive KB cell (Fig.2). Moreover, the large number of minicells uptake in LNCaP cells could be

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attributed to the PSMA (prostate specific membrane antigen) which are uniquely overexpressed by these cells [33]. FA

minicellsVEGFA endocytosis was also evaluated in this study. Receptor

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Mechanism of

mediated endocytosis occurs at 37°C and stops at 4°C. Furthermore, receptor mediated endocytosis do not occurs without the specific ligand of that receptor. Additionally, in the presence of free ligand, competition arise between ligand conjugated nanoparticle and free ligand for the uptake. In this study, folic acid conjugated minicells were not taken up by cells at 4°C. Similar result was observed when minicells were delivered without targeting moiety. Uptake of folic acid conjugated minicells reduced in presence of free folic acid. These results suggested that minicells were taken up by folate receptor overexpressing cells by receptor mediated endocytosis. Furthermore, it also confirmed that active targeting by folic acid is required for the internalization of minicells.

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ACCEPTED MANUSCRIPT It has been known that cells can internalize materials by several endocytotic mechanisms such as clathrin-mediated endocytosis, caveolae-mediated endocytosis, phagocytosis and macropinocytosis [34, 35]. Sodium azide, which blocks cellular ATP synthesis, resulted in a

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significant decrease in the cellular internalization indicating that internalization of FA

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minicellsVEGFA is an energy-dependent process. Phenylarsine oxide is an inhibitor of the

clathrin mediated endocytosis, which is most related to specific receptor ligand interaction [36]. Weaker fluorescence was observed when FITC loaded FAminicellsVEGFA were delivered

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in the presence of phenylarsine oxide (fig 3 e, f), therefore clathrin mediated endocytosis was involved in its uptake mechanisms. Caveolae mediated endocytosis is another major pathway

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for cellular uptake, which can be inhibited by filipin. This kind of endocytosis process is associated with the surface charge of the nanoparticles, which turns to be adsorption mediated endocytosis [37]. The cellular internalization of FAminicellsVEGFA were not inhibited by filipin (fig. 3 e, f), suggesting that the caveolae-mediated endocytosis process was not responsible for the uptake of

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minicellsVEGFA by cells. Endocytosis through macropinocytosis can be

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inhibited by colchicine, is associated with the particle size of nanoparticle. Treatment with colchicine did not reduce the uptake of FA

FA

minicellsVEGFA in cells. Hence, uptake of

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minicellsVEGFA was found to be energy dependent and clathrin-mediated endocytosis which

is in conformity with previously described folate conjugated fluorescent silica nanoparticles

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[29].

Subsequently, qualitative gene expression was analyzed following in vitro delivery of FA

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minicellspRNAT. A549 cells showed very low level of folate receptor expression; so folic

acid conjugated minicells seldom delivered pRNAT into these cells. Contrary, KB and LNCaP showed large number of GFP positive cells per field (fig. 4). Hence, from this experiment it could be inferred that after endocytosis, folic acid conjugated minicells passed through the well-established endosomal pathways. Subsequent endosomal escape delivered packaged expression vector which later on expressed the transgene. Similarly, in vitro delivery of

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minicellsVEGFA suggested that uptake of

folate and PSMA receptor.

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minicellsVEGFA were dependent on

FA

minicellsVEGFA rarely delivered VEGFA shRNA in A549 cells

due to the low level of folate receptor expression. The delivery of scramble did not change the expression of VEGFA in any of the selected cell lines (fig. 5). This indicated that active shRNA must be delivered to silence the targeted gene.

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minicellsVEGFA has failed to lessen the tumor volume in A549 xenograft which is in

agreement with the in vitro studies.

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minicellsVEGFA has significantly reduced the tumor

volume in KB and LNCaP xenograft (fig. 6). Reduction in the tumor cannot be due the

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probable activation of TLR-4 and subsequent inflammatory response by minicell cell wall or

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endotoxin because none of the other treatment like non-targeted or non-specifically targeted minicells hindered the tumor growth. Additionally, diminished vascularisation was also FA

recorded in

minicellsVEGFA treated KB and LNCaP xenograft (Supplementary fig. 5).

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Moreover, PCR revealed that the gene expression of VEGFA was significantly downregulated in these xenografts (fig. 7). Further, there was no significant difference in the

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levels of IFN-γ, TNF-α and IL-6 in mice treated with FA

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minicellsVEGFA, compared with

minicellsScramble (Supplementary fig. 6). Hence, tumor regression can be attributed to

inhibition of angiogenesis through knock-down of VEGFA by

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minicellsVEGFA.

Furthermore, folic acid conjugated minicells were found to be well tolerated because none of the injected mice showed any adverse reaction throughout the treatment. Mice body mass

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graph is provided as supplementary fig. 4. FA

minicellsVEGFA showed that majority of

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minicellsVEGFA

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Finally, in vivo biodistribution of

were distributed in tumor tissue which was due to both passive and active targeting of FA

majority of Secondly,

FA

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minicellsVEGFA (fig. 8). Tumor formed more poor and leaky blood vessels through which minicellsVEGFA diffused into the tumor microenvironment by passive targeting.

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minicellsVEGFA were actively targeted to tumor cell surface folate receptor by

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folic acid, followed by receptor engagement and endocytosis. Fluorescence observed in other organ like liver, heart and spleen was found to be in blood vessels. Expression of folate receptor is absent in most normal tissue, so

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minicellsVEGFA would not retained in normal

tissue and quickly washed out. Moreover, size of folic acid conjugated minicells is large enough which prevented crossing of blood brain barrier. Furthermore, large size also prevented minicells to be filtered through the glomerulus in the kidney [38]. Hence, it avoided non specific targeting of shRNA into other vital organs.

In this way, folic acid conjugated minicells offers a great potential for in vivo targeted delivery of shRNA to cancer cells. In vitro and in vivo studies collectively corroborate that, FA

minicellsVEGFA had efficiently delivered packaged shRNA to variety of targeted cancer

cells. Moreover, after delivery, active shRNA had successfully silenced the VEGFA mRNA, 17

ACCEPTED MANUSCRIPT both in cell lines as well as in tumor xenograft. Extensive evaluation of pro-inflammatory cytokines and interferons followed by long term

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minicellsVEGFA administration will be

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required to determine its immunogenicity.

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ACCEPTED MANUSCRIPT Figure captions

Fig.1. Characterization of minicells producing bacterial strain (E.coli PB114), (a) Light

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packaging of shRNA, minicells purification and folic acid conjugation.

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microscopy image of E.coli PB114 after gram’s staining (1000 X magnification), (b) Transmission electron microscopy image of E.coli PB114. (20000X magnification). Fig. 1(b) (i), (ii) and (iii) shows sequential minicell production from the parent E.coli PB114 which is

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indicated by arrows. Scale bars 500 nm, 1000 nm and 1000 nm, respectively. (c) Fluorescence microscopy image of purified minicells loaded with FITC (1000 X

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magnification), (d) Transmission electron microscopy image of purified minicells (20000 X magnification) Scale bar 1000 nm, (e) Gel electrophoresis of isolated VEGFA shRNA plasmid and pSUPERneoScramble from minicells. Gel showing A) 1) Isolated VEGFA shRNA plasmid from minicells 2) Purified VEGFA shRNA plasmid, B) 1) Isolated pSUPERneoScramble from minicells 2) Purified pSUPERneoScramble. Bands of isolated

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plasmid DNA corresponds to standard purified plasmids which confirmed the presence of VEGFA shRNA plasmid and pSUPERneoScramble plasmid into the minicells.

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(f) Schematic representation of folic acid conjugation with shRNA packaged minicells, (g) Minicell pellet. A) Before folic acid conjugation reaction and, B) After folic acid conjugation

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reaction. Yellow colour of minicell pellet indicates successful folic acid conjugation. (Minicell pellet indicated with arrow), (h) TEM images before and after folic acid conjugation. TEM images A) before folic acid conjugation B) after folic acid conjugation

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(20000X magnification, minicells are indicated with arrows) Scale bar 1000 nm and 400 nm, respectively, (i) Graph showing average size of folic acid conjugated minicells.

Fig.2. In vitro delivery of folic acid conjugated minicells. Microscopy image of (a) A549, (b) KB, and (c) LNCaP cells after delivery of (i) 1010, (ii) 109, (iii)108, (iv)107, (v)106 folic acid conjugated minicells, A) Fluorescence image B) Phase contrast image (200X magnification).

Fig.3. Mechanism of minicell uptake and endocytosis. Microscopy image of (a) KB and (b) LNCaP cells after delivery of FITC loaded minicells (i) minicellsVEGFA at 37°C, (iii)

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minicellsVEGFA at 4°C, (iv)

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minicellsVEGFA at 37°C, (ii)

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minicellsVEGFA with excess of free

folic acid at 37°C, A) Fluorescence image B) Phase contrast image (200X magnification), 22

ACCEPTED MANUSCRIPT Graph showing quantitative minicells uptake in terms of mean fluorescence /mg of protein in different groups (as indicated above) of (c) KB cells and (d) LNCaP cells. Fluorescence was represented as mean±SD values from at least three different experiments

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Microscopy image of (e) KB and (f) LNCaP cells after delivery of FITC loaded minicells

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(FAminicellsVEGFA) in the presence of (i) sodium azide (0.1%), (ii) colchicine (2 µg/mL), (iii) filipin complex (4 µg/mL), and (iv) phenylarsine oxide (0.5 µg/mL), A) Fluorescence image

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B) Phase contrast image (200X magnification), Graph showing quantitative minicells endocytosis in terms of mean fluorescence /mg of protein in different groups (as indicated

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above) of (g) KB cells and (h) LNCaP cells. Fluorescence was represented as mean±SD values from at least three different experiments. FA

Fig.4. In vitro delivery of

minicellspRNAT and gene expression analysis. In vitro gene

expression in (i) A549, (ii) KB, and (iii) LNCaP cells after

FA

minicellspRNAT delivery. A)

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Fluorescence image B) Phase contrast image (100X magnification). Cells expressing GFP are indicated with arrows for A549 cells. FA

minicellsVEGFA and gene expression analysis. PCR and gene

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Fig.5. In vitro delivery of

expression analysis of VEGF A from (a) A549, (b) KB, and (c) LNCaP cells after in vitro FA

minicellsVEGFA; A) Cropped gel picture of amplified PCR products from

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delivery of

respective cells 1) FAminicellsscramble, 2) minicellsVEGFA, 3) FAminicellsVEGFA, B) Relative gene

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expression of VEGFA, normalized with GAPDH, C) Relative gene expression of VEGFA normalized with 18s. Here, G-1 to G-3 indicates treatment with minicellsVEGFA, and

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minicellsscramble,,

FA

minicellsVEGFA, respectively. Statistically significant when compared

with *FAminicellsScramble, #minicellsVEGFA, p<0.05. Fig.6. In vivo delivery of

FA

minicellsVEGFA and mean tumor volume. Mice bearing tumor

xenograft of (a) A549, (b) KB, and (c) LNCaP cells. A) At start and B) end of the treatment with i) saline, ii) FAminicellsScramble iii) minicellsVEGFA, iv) and FAminicellsVEGFA. Locations of tumors are indicated with arrows. Graphs showing mean tumor volume of d) A549, e) KB, and f) LNCaP xenograft from different groups at every week till the end of the treatment (n=6). Statistically significant when compared with *saline, #FAminicellsscramble, and ΨminicellsVEGFA, p<0.05.

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ACCEPTED MANUSCRIPT Fig.7. In vivo gene expression of VEGFA by PCR. PCR and gene expression analysis of VEGF A from (a) A549, (b) KB and (c) LNCaP xenograft after in vivo delivery of FA

minicellsVEGFA A) Cropped gel picture of amplified PCR products from respective tumor FA

minicellsVEGFA. B) Relative

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tissue 1) Saline, 2) FAminicellsscramble, 3) minicellsVEGFA, and 4)

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gene expression of VEGFA normalized with GAPDH, C) Relative gene expression of VEGF A normalized with 18s. Here, G-1 to G-4 indicates treatment with saline, minicellsVEGFA, and

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minicellsscramble,

minicellsVEGFA, respectively. Statistically significant when compared

Fig.8. In vivo biodistribution study of

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with *saline, # FAminicellsScramble, and ΨminicellsVEGFA, p<0.05.

minicellsVEGFA.. Microscopy image, A)

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fluorescence image B) light miccrosopy of H and E stained cryosection of (i) tumor, (ii) liver, (iii) lung, (iv) kidney, (v) spleen, (vi) heart, (vii) brain, and (viii) skin after in vivo delivery of

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FITC loaded FAminicellsVEGFA (100X Magnification).

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Figure 3a and b

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Figure 3c and d

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Figure 3e and f

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Figure 3 g and h

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ACCEPTED MANUSCRIPT Table 1: Primer sequences Forward Primer

Reverse Primer

Amplicon

VEGFA

5’-

5’-

102

TCTTCAAGCCATCCT

TCTGCATGGTGATGT

GTGTG-3’

TGGAC-3’

5’-

5’-

CATGAGAAGTATGAC

AGTCCTTCCACGATA

AACAGCCT-3’

CCAAAGT-3’

5’-

5’-

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GTAACCCGTTGAACC

CCATCCAATCGGTAG

CCATT-3’

TAGCG-3’

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18s

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GAPDH

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Gene

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113

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