Enhanced Secretion of Functional Insulin with DNA-Functionalized

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Enhanced Secretion of Functional Insulin with DNA-Functionalized Gold Nanoparticles in Cells Kian Ping Chan, Sheng-Hao Chao, and James Chen Yong Kah ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.9b00032 • Publication Date (Web): 04 Feb 2019 Downloaded from http://pubs.acs.org on February 4, 2019

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ACS Biomaterials Science & Engineering

Enhanced Secretion of Functional Insulin with DNA-Functionalized Gold Nanoparticles in Cells Kian Ping Chan1,2,3, Sheng-Hao Chao2,4*, James Chen Yong Kah1,5*

1NUS

Graduate School for Integrative Sciences and Engineering, Centre for Life

Sciences (CeLS), #05-01, 28 Medical Drive, Singapore 117456

2Bioprocessing

Technology Institute, Agency for Science, Technology and Research,

Singapore, 20 Biopolis Way, #06-01 Centros, Singapore 138668

3Present

Address: NanoBio Lab, 31 Biopolis Way, #09-01 The Nanos, Singapore

138669.

4Department

of Microbiology and Immunology, National University of Singapore, 5

Science Drive 2, Blk MD4, Level 3, Singapore 117597

1 ACS Paragon Plus Environment

ACS Biomaterials Science & Engineering 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

5Department

of Biomedical Engineering, National University of Singapore, 4

Engineering Drive 3, Blk E4, #04-08, Singapore 117583

Corresponding Author *[email protected] *[email protected]

KEYWORDS

Gold nanoparticles, DNA oligomers, mRNA translation, protein corona, insulin

ABSTRACT

We have previously shown the use of gold nanoparticles (AuNPs) functionalized with DNA (AuNP-DNA) to increase insulin mRNA translation in a cell-free system. In this study, we translate the concept into a whole cell system to demonstrate functionality despite the additional complexity of intracellular delivery and mRNA translation inside living cells. We selected an insulin-secreting pancreatic islet cell line, RIN-5F, as our model and designed a DNA oligomer (insDNA) that is complementary to the 3’-


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untranslated region of insulin mRNA for conjugation to AuNPs (AuNP-insDNA). AuNPinsDNA was stable in the extracellular environment of RIN-5F cells up to 24 h, without eliciting any cell toxicity. Upon cellular entry, AuNP-insDNA was able to sustain enhanced insulin secretion from 6 h to 12 h post-incubation, peaking at 10 h with an enhancement factor of 1.69-fold. This enhancement was not observed when insDNA was removed or replaced with poly-thymine or poly-adenine DNAs. The enhanced insulin secreted was 100% functional and capable of binding to its insulin receptor. The outcome of this study demonstrated the feasibility of AuNP-DNA to enhance the synthesis of proteins in whole cells and could serve as a new direction of invoking a patient’s own beta cells to increase insulin secretion for treatment of diabetes.



INTRODUCTION

Type 2 Diabetes Mellitus is commonly characterized by high blood glucose levels in patients due to insufficient level of blood insulin, a hormone that regulates glucose metabolism. Besides the standard care of ingesting or injecting exogenous insulin, nanocarriers have also been developed to improve insulin delivery to the target site1-2, with controlled release of insulin for prolonged treatment duration. However, insulin delivery is still limited by its reservoir amount. Cell-based therapy could enable prolonged insulin supply but suffers from allogenic rejection by the immune system. Although transplanted beta cells could be protected by a layer-by-layer nanothin polymer coating while still permitting diffusion of necessary nutrients such as glucose, oxygen and insulin3-4, they still suffer from the undesirable yet unavoidable formation of protein corona coat in any protein-rich biological media5-11, which limits the intended surface functionality12-13, insulin release from nanocarriers, or isolates transplanted beta cells from the biological environment.


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Various strategies have been developed to minimize protein corona formation using zwitterionic-coatings14-15 or polyethylene glycol (PEG) coating16-20. We have previously embraced the protein corona into our design of nanoparticles for various bioapplications21-25, including a novel DNA functionalized gold nanoparticles (AuNPs) that enhanced the translation of mRNA in a cell-free HeLa lysate system26-27. The concept worked through a dual mechanism of having the DNA oligomer to selectively recruit the mRNA of interest through DNA/RNA hybridization while the surface of AuNPs non-specifically adsorb ribosomes and other translation proteins to form a protein corona and create a focal point of high concentration of the translation machineries to enhance the mRNA translation efficiency. Here, AuNPs were used for their ease of conjugation to DNA oligomers28-30, good cellular uptake31 and biocompatibility29, 32. In this study, we extend this concept to enhance insulin secretion in a pancreatic islet RIN-5F cell line model and demonstrate the feasibility of translating our AuNP-DNA technology from a cell-free to a cellular environment with a higher level complexity involving cellular uptake, possible endosomal escape, and multiplex of competing mRNAs in the living cell (Figure 1). Furthermore, our present study also presented a 5 ACS Paragon Plus Environment


kinetic dimension to the enhanced insulin secretion, and evaluated the functionality of the secreted insulin which was not reported in our cell-free system. Here, the translation into a whole cell system also represents novelty in itself since the use of nanotechnology to enhance insulin synthesis in living cells has not been demonstrated to date, to our knowledge. This has important implications in bioprocessing optimization approaches, and the development of nanotechnology for treatment of Type 2 Diabetes Mellitus. The complementary binding of DNA oligomers (insDNA) functionalized on AuNPs (AuNP-insDNA) to the 3’-UTR of insulin mRNA, coupled with the non-specific adsorption of intracellular proteins effected a maximum 1.69-fold increase in insulin secretion after 10 h of RIN-5F cells exposure to AuNP-insDNA, which was not observed in our controls of poly(A) and poly(T) DNA oligomers. Our AuNP system represents a novel use of nanotechnology away from their conventional role as insulin nanocarriers towards one which modulates the insulin production in cells while exploiting the “undesirable” protein corona to do so.


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Figure 1. Schematic showing the application of AuNP-insDNA to enhance insulin production in pancreatic islet RIN-5F cells. (i) AuNP-DNA remained colloidally stable in extracellular environment of RIN-5F cells, while citrate-capped AuNPs aggregated. The stable AuNP-DNA were (ii) taken up into cells and (iii) translation of insulin mRNA was enhanced through recruitment of insulin mRNA and other translation proteins to create a high focal concentration of these translation machineries on AuNP-insDNA. (iv) The



insulin produced at enhanced levels remained functional and capable of binding to its receptor.

MATERIALS AND METHODS

Synthesis and Characterization of AuNPs AuNPs were synthesized using a previously published protocol33. The synthesized citrate-capped AuNPs were cooled to room temperature before performing repeated centrifugation at 9,000 rpm for 20 min to remove excess citrate. Finally, the pellet was reconstituted in nuclease-free water and stored at 4 °C before use. The optical properties of AuNPs were characterized using UV-Vis spectrometry (UV-2450, Shimadzu, Japan) to determine its peak surface plasmon resonance wavelength and concentration34. Additionally, the dimension of AuNPs were measured using transmission electron microscopy (TEM) (JEM-1220, JEOL Ltd., Japan) and Zetasizer (Nano ZS, Malvern, UK) to give its core size and hydrodynamic diameter (DH) respectively. The zeta potential (ζ) of AuNPs was also measured using Zetasizer.


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AuNP-DNA Conjugation DNA oligomers of 25 nucleotides were purchased from Integrated DNA Technologies (USA) and had a modified 5’-thiol end to facilitate covalent conjugation onto surface of AuNPs. Since the 3’-UTR region was shown previously to be suitable candidate for DNA hybridization, one set of DNA was designed to hybridize to 3’-UTR of insulin mRNA (insDNA: 5’-TTTTT TTTTT GGTGC TCATT CAAAG-3’) while having the lowest homology to other cellular mRNAs to avoid non-specific hybridization. Two sets of DNA oligomers were also designed to be non-insulin hybridizing negative controls (p(T)DNA: 5’-TTTTT TTTTT TTTTT TTTTT TTTTT-3’; p(A)DNA: 5’-AAAAA AAAAA AAAAA AAAAA AAAAA-3’). Conjugation of DNA to AuNPs was performed based on our previous published work26-27, optimized from a published protocol29, 35. A low pH assisted method reduced conjugation time by protonation of adenines to lower the overall negative charge of DNA oligomers and thus reduce their repulsion with the negatively-charged surface of AuNPs36. AuNP-DNA was subsequently purified by repeated centrifugation at 11,000 9 ACS Paragon Plus Environment


rpm for 30 min and reconstituted in nuclease-free water to remove unbound DNA before use. The optical properties, hydrodynamic diameter and zeta potential of AuNP-DNA were characterized similarly to synthesized citrate-capped AuNPs. The number of bound DNA per AuNP was determined by subtracting the amount of unbound DNA, collected from centrifugal washes, from the initial amount of DNA added to the AuNPs. Quantitative measurement of DNA concentration was performed by using SYBR Gold nucleic acid stain (Thermo Fisher Scientific, USA) which intercalates DNA and its fluorescence measured against calibrating standards of known DNA concentrations using Infinite 200 PRO (Tecan, Switzerland).

Cell Culture Rat pancreatic beta cells, RIN-5F (ATCC, USA) was maintained in RPMI media (Thermo Fisher Scientific, USA) with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, USA) and 1 % Penicillin-Streptomycin-Glutamine (PSG) (Thermo Fisher Scientific, USA) at 37 °C in a humidified environment with 5% CO2. 10 ACS Paragon Plus Environment

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Colloidal Stability of AuNP-DNA The colloidal stability of AuNP-DNA in the extracellular environment of RIN-5F cells was determined based on the optical properties of AuNPs. Briefly, 1 × 105 RIN-5F cells suspended in 1 ml of RPMI media was cultured in each well of a 24-well plate for three days before addition of 100 µl of 50 nM citrate-capped AuNPs, AuNP-p(A)DNA, AuNPp(T)DNA or AuNP-insDNA to the cell culture. 100 µl of RPMI containing AuNPs or AuNP-DNA and cell-secreted proteins was collected from each well at 2 h interval from 0 h to 12 h. Subsequently, each samples collected were loaded into a 96-well plate and their UV-Vis spectrum was acquired using UV−Vis absorption spectroscopy to assess their colloidal stability.

AuNP-DNA Uptake To determine the uptake of citrate-capped AuNPs and AuNP-DNAs in RIN-5F cells, 1 × 104 RIN-5F cells were first suspended in 100 µl of RPMI media and cultured in a 96well plate for 3 days to allow RIN-5F cells to adhere strongly to the bottom of the well. 11 ACS Paragon Plus Environment


Following this, 10 µl of 50 nM AuNPs or AuNP-DNA was added in triplicates to the RIN5F cells and incubated for 0 h, 4 h, 6 h, 8 h and 12 h. At each time point, cells were rinsed twice with 1× phosphate buffered saline (PBS) before adding 100 µl of trypsinEDTA (0.25%) (Thermo Fisher Scientific, USA) for 10 min to dislodge the cells from the cell culture plate. 90 µl of trypsinized cells was then added with 300 µl of aqua regia (final concentration of acid: 1.5% (v/v) HNO3 and 4.5% (v/v) HCl) for 30 min. 300 µl of mixture was subsequently diluted with 4.7 ml of ultra-pure water in a inductively coupled plasma mass spectrometry (ICP-MS) tube before performing ICP-MS (7700x ICP-MS, Agilent Technologies, USA) according to manufacturer’s protocol. Microscopy images of RIN-5F cells dosed with AuNPs or AuNP-DNA were also acquired to show the uptake of AuNPs inside cells. Briefly, 1 × 105 RIN-5F cells were seeded inside Nunc™ Lab-Tek™ II CC2™ Chamber Slide System (Thermo Fisher Scientific, USA) for 4 days. Each chamber was then washed twice with 1× PBS before adding 500 µl of 10 % formalin solution, neutral buffered, (Sigma-Aldrich, USA) for 10 min at room temperature to fix the cells. Following cells fixation, each chamber was washed thrice with 1× PBS before adding 17 µl of VECTASHIELD Antifade Mounting 12 ACS Paragon Plus Environment

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Medium

(Vector

Laboratories,

USA)

containing

4′,6-diamidino-2-phenylindole

dihydrochloride (DAPI) to stain the nucleus. Dark field and fluorescence images were obtained using a Nikon Ci-L Fluorescence Upright microscope (Nikon Instruments, Japan), which is equipped with a CytoViva 150 Condenser (CytoViva, USA) and an sCMOS camera (pco.edge 4.2 M-USB-PCO, PCO, Germany). An oil immersion 60× objective was used in the imaging.

Cell Viability of AuNP-DNA Potential cytotoxicity of AuNP-DNAs on RIN-5F cells was investigated by measuring cell viability of RIN-5F after AuNP-DNAs incubation. Similar to our cell uptake studies, 1 × 105 RIN-5F cells suspended in 1 ml of RPMI media was cultured in each well of a 24well plate for three days before addition of 100 µl of 50 nM AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA or AuNP-insDNA to the cells and incubations at 37 °C over the next 24 h. After incubation, cell media containing AuNPs or AuNP-DNAs was removed and the cells were rinsed twice with 1 ml of 1× PBS and resuspended in 1 ml of fresh RPMI for 24 h. The final cell viability was assessed by adding 100 µl of 10× PrestoBlue® cell 13 ACS Paragon Plus Environment


viability reagent (Thermo Fisher Scientific, USA) to each well and incubated at 37 °C for 1 h in the dark before fluorescence measurement (Ex: 560 nm, Em: 590 nm) to determine the amount of live cells.

Enhancement of Insulin Secretion Quantitative assessment of insulin secretion from RIN-5F was conducted to determine if AuNP-insDNA was able to enhanced insulin mRNA translation inside RIN-5F cells. 1 × 105 RIN-5F cells suspended in 1 ml of RPMI media was cultured in each well of a 24well plate for three days before addition of 100 µl of 50 nM AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA or AuNP-insDNA to modulate the insulin mRNA translation. 100 µl of cell culture media containing non-uptaken AuNPs or AuNP-DNAs and cell-secreted proteins was collected from each well at 2 h interval from 0 h to 12 h for insulin quantification. The collected samples were diluted with 100 µl of 1× Assay Diluent B provided by Insulin Rat ELISA kit (Thermo Fisher Scientific, USA) before proceeding accordingly to manufacturer’s protocol for ELISA quantification of the protein.


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In a separate experiment to find the minimal dose of AuNP-insDNA needed for translation enhancement, the same volume of 10 nM or 30 nM of AuNPs, AuNPp(A)DNA, AuNP-p(T)DNA and AuNP-insDNA was added instead of 50 nM. Their cell culture supernatant was collected for ELISA quantification of secreted insulin at 8 h post-incubation, when the insulin secretion enhancement for 50 nM AuNP-insDNA started to saturate.

Functional Assay of Insulin The functional conformation of secreted insulin produced by enhanced mRNA translation using AuNP-insDNA was examined using PathHunter® Insulin Bioassay kit (Eurofins DiscoverX Corporation, USA). To first stimulate the secretion of insulin, 1 × 105 RIN-5F cells suspended in 1 ml of RPMI media was cultured in each well of a 24well plate for three days before addition of 100 µl of 50 nM AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA or AuNP-insDNA to the RIN-5F cells. 100 µl of cell culture media containing non-uptaken AuNPs or AuNP-DNAs and cell-secreted proteins was collected from each well at 2 h interval from 0 h to 12 h. All samples collected were centrifuged at 15 ACS Paragon Plus Environment


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11,000 rpm for 30 min to remove AuNPs or AuNP-DNA. 20 µl of the supernatant was used subsequently for the insulin bioassay kit according to manufacturer’s protocol. The percentage of functional insulin at any given time was calculated based on the following equation (1): % 𝐹𝐼𝐴𝑢𝑁𝑃 ― 𝐷𝑁𝐴 =

𝐴𝑏𝑠. 𝐹𝐼𝐴𝑢𝑁𝑃 ― 𝐷𝑁𝐴 𝐴𝑏𝑠. 𝑇𝐼𝐴𝑢𝑁𝑃 ― 𝐷𝑁𝐴

𝐴𝑏𝑠. 𝐹𝐼𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒

÷ 𝐴𝑏𝑠.

𝑇𝐼𝑏𝑎𝑠𝑒𝑙𝑖𝑛𝑒

× 100% ,

(1)

where FI = Functional Insulin; TI = Total Insulin The ratio of functional insulin to total insulin produced with AuNP-DNAs dosed to the RIN-5F cells was normalized to the ratio of baseline functional insulin secretion to baseline total insulin secretion by RIN-5F alone to account for variables introduced when moving from ELISA kit to bioassay kit.

RESULTS AND DISCUSSION

Characterization of DNA conjugated AuNPs The synthesized citrate-capped AuNPs were isolated and monodispersed with an average diameter of 12.34 ± 0.09 nm as analyzed from TEM images (Figure 2a,b). UV-


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Vis absorption spectrum of AuNPs showed a peak at 519 nm and was observed to redshift to 528 nm, 523 nm and 525 nm after conjugation to p(A)DNA, p(T)DNA and insDNA oligomers respectively (Figure 2c) due to the ligand replacement from citratecapped to DNA-conjugated surface, which resulted in a change in local refractive index37-38. In addition, there were minimal peak broadening observed for all AuNP-DNA constructs, signifying that AuNP-DNA retained colloidal stability after DNA conjugation.



Figure 2. Characterization of synthesized citrate-capped AuNPs and three types of DNA conjugates; AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA used in this study. (a) TEM image of synthesized citrate-capped AuNPs and (b) histogram of its size distribution. (c) UV-Vis spectrum of AuNPs showed a defined peak at 519 nm with a red-shift in peak upon DNA conjugation. (d) Hydrodynamic diameter (DH) distribution of AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA determined by dynamic light scattering (DLS) indicated a small increment in DH upon DNA conjugation without forming large aggregates. (e) Zeta potential, ζ, was negative for both AuNPs and AuNPDNA due to coating of negatively-charged citrate and DNA respectively. (f) All AuNPDNAs had approximately 80 DNA strands per AuNP after conjugation.

There was also a shift to larger hydrodynamic diameter (DH) in the size histogram of AuNPs as measured by dynamic light scattering (DLS) upon DNA conjugation (Figure 2d). This was attributed to the DNA oligomers of 25 nucleotides extending outward from the surface of AuNPs to increase the DH of AuNP-DNA which was not picked up by the


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TEM. The absence of large aggregates as indicated by the monomodal peak in the size histogram further demonstrated colloidal stability of AuNP-DNA in nuclease-free water. Such a good colloidal stability was conferred by charge stabilization as the zeta potential (ζ) measurements of AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA and AuNPinsDNA were all negative at -30.87 ± 4.48 mV, -31.70 ± 2.51, -26.40 ± 1.83 mV and 34.90 ± 0.71 mV respectively (Figure 2e), due to the presence of negatively-charged citrate or DNA. Our conjugation protocol also loaded an average of 80.8 ± 1.3, 84.7 ± 1.93 and 81.0 ± 1.29 DNA oligomers on each AuNP for AuNP-p(A), AuNP-p(T)DNA and AuNP-insDNA respectively with insignificant difference between them (one-way analysis of variance, (F(2,14) = 1.602, p = 0.2417)) (Figure 2f). The DNA density on our AuNPs was similar to what was reported in literature39.

Stability of AuNP-DNA in RIN-5F Extracellular Environment The colloidal stability of AuNP-DNA in an extracellular environment of cell media containing serum proteins and secretion products from cells is critical for their 19 ACS Paragon Plus Environment


functionality as aggregation would mask DNA oligomers from their surface and consequently hinder their hybridization to mRNA for translation enhancement. In the absence of DNA-capping, the citrate-capped AuNPs aggregated in cell media as indicated by the gradual redshift and peak broadening in the UV-Vis absorption spectrum after 2 h incubation in RIN-5F extracellular environment, which continued up to 24 h (Figure 3a). The high ionic concentration in cell media provided charge screening to reduce electrostatic repulsion between AuNPs, thus bringing them close enough to form irreversible aggregates.


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Figure 3. Stability of citrate-capped AuNPs and AuNP-DNA in the RIN-5F extracellular environment. (a) UV-Vis spectrum showed progressively peak broadening of citratecapped AuNPs over time indicating gradual aggregation of the AuNPs. On the other hand, a distinct UV-Vis peak with no significant peak broadening was maintained over time for (b) AuNP-p(A)DNA, (c) AuNP-p(T)DNA and (d) AuNP-insDNA.

In contrast, all three AuNP-DNAs maintained their colloidal stability with minimal redshift and peak broadening up to 24 h post-incubation in the RIN-5F extracellular environment (Figure 3b-d). Here, the conjugated DNA provided a layer of steric stabilization and prevented the aggregation of AuNP-DNA even in high ionic concentrations. The stability of AuNP-DNA further affirmed successful DNA conjugation and that the approximate 80 DNA strands conjugated on each AuNP were sufficient to confer colloidal stability in the extracellular environment of RIN-5F cells.

Uptake of AuNP-DNA into RIN-5F cells



Before AuNP-DNA could enhance the translation of insulin mRNA in cells, it has to first pass through the cell membrane and enter the cytoplasm where mRNA translation occurs. By quantifying cell uptake of AuNPs at various time points using ICP-MS, we observed an increasing cellular uptake of citrate-capped AuNPs in RIN-5F from 4 h to 12 h, with intracellular AuNPs amount peaking at (3.40 ± 0.40) × 106 particles per cell. This amount was much higher (~10-folds higher) than the uptake of AuNP-DNAs in general, where the intracellular AuNPs amount seemed to saturate after 4 h with an average of (2.15 ± 0.07) × 105, (2.38 ± 0.05 ) × 105 and (2.57 ± 0.12) × 105 particles per cell for AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA respectively (Figure 4a). These corresponded to ~1% of total AuNP-DNA dosed to the RIN-5F cells. The higher uptake of citrate-capped AuNPs was not unexpected given that they aggregated severely in cell media before cell entry as shown by the redshift and peak broadening in their absorbance spectra (Figure 3a). Aggregated AuNPs have been shown to exhibit higher uptake in cells compared to colloidally stable AuNPs40-41 such as the AuNP-DNAs used in our study. Furthermore, the strong negative surface charge


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on AuNP-DNAs conferred by the DNA interface also minimized their interaction with the negatively charged cell membrane, which in turn hindered their uptake.

Figure 4. Cellular uptake and toxicity of AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA. (a) Citrate-capped AuNPs showed a progressive increased in their amount in RIN-5F cells with incubation time while a non-increasing amount of AuNPp(A)DNA, AuNP-p(T)DNA and AuNP-insDNA was observed from 4 h to 12 h. (b) Microscopy images of RIN-5F cells alone and dosed with AuNPs or AuNP-DNA for 6 h were captured. AuNPs were visualized under dark field imaging as bright spots due to



their strong optical scattering. They were found to surround cell’s nucleus, which was stained with DAPI. All scale bars are 50 µm. (c) 100% cell viability was observed when RIN-5F cells were incubated with AuNPs or AuNP-DNA for 24 h.

Despite the lower cellular uptake of AuNP-DNAs compared to citrate-capped AuNPs, the amount was still sufficient to elicit an enhanced translation as described later. At this point, we would like to highlight that our cell uptake was reported based on the absolute number of particles uptaken per cell instead of the percentage relative to the amount of AuNP-DNA dosed. Here, the purpose of our uptake study was not to compare the uptake between different DNA functionalization, but to demonstrate similar level of intracellular uptake between AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA in RIN-5F cells to eliminate any AuNP concentration-dependent mRNA translation arising from differing numbers of AuNP-DNA in cells, thus dismissing differential AuNP-DNA uptake as a confounding factor in subsequent translation enhancement in cells.


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The cellular uptake of AuNPs in RIN-5F cells was further confirmed with dark-field imaging where the strong optical scattering of AuNPs appeared as bright scattering signal which was most clearly observed in RIN-5F cells dosed with citrate-capped AuNPs after 6 h. On the other hand, the signal intensity from AuNP-p(A)DNA, AuNPp(T)DNA and AuNP-insDNA were less intense compared to citrate-capped AuNPs, but similar between AuNP-DNAs which agreed with the quantitative measurements from ICP-MS (Figure 4b). The merged fluorescent and dark-field images also showed little overlap between the scattering signal from AuNPs and the fluorescence from DAPI, thus suggesting that the AuNPs were localized in the cytoplasm and not the nucleus, which is critical since mRNA translation occurs in the cytoplasm. We also noted that the citrate-capped AuNPs and AuNP-DNAs dosed to RIN-5F cells maintained 100 % cell viability after 24 h (Figure 4c), suggesting their biocompatibility and bioinertness in cells. This also meant that the difference in cell uptake between citrate-capped AuNPs and AuNP-DNAs was not attributed to their viability which could have compromised the cell uptake.



Increased Insulin Secretion The AuNP-DNAs taken up by RIN-5F cells would exert its role to recruit both mRNAs and proteins related to the translation machineries e.g. ribosomes, elongation factors and translation factors through non-specific adsorption to form the protein corona around AuNPs. Here, we proposed that the DNA oligomers hybridized to their respective mRNAs at their 3’-UTR to create a focal point of high mRNA concentration where all the translation machineries were brought into close proximity of each other through non-specific adsorption on the surface of AuNPs to facilitate efficient translation, with a similar mechanisms described in details in our previous lysate studies26-27. In this study, we deliberately designed the insDNA to hybridize with the 3’-UTR of insulin mRNA. In our previous study, we showed that hybridizing the 3’-UTR region of the target gene may minimize the interference with normal processes of mRNA translation and enhance the protein synthesis26. To minimize the non-specific effects, we designed the insDNA with the lowest cross homology to other cellular genes. In contrast, our poly(A)DNA and poly(T)DNA served as non-3’-UTR hybridizing controls. 26 ACS Paragon Plus Environment

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Here, p(T)DNA was selected as it could non-specifically hybridize to long poly(A)-tail found in any mRNAs to serve as our non-insulin specific control while p(A)DNA was selected as it should not hybridize to 3’-UTR of insulin mRNA. Free insDNA was not included in our study due to the constraint in ensuring equivalent delivery of short insDNA oligomers at the same amount as those conjugated on AuNPs, as well as their instability due to potential nuclease degradation in cells. Furthermore, previous studies by others have also shown that free DNA in cell-free lysate systems did not exhibit any translation enhancement7. Using enzyme-linked immunosorbent assay (ELISA) to quantify the amount of secreted insulin in the extracellular media of RIN-5F cell culture and by normalizing to the baseline insulin secretion from RIN-5F cells, we observed a gradual increase in the enhancement of insulin secretion with AuNP-insDNA dosing over a 10 h period which peaked at 1.69-fold enhancement against our normalized baseline secretion level (Figure 5a,b). Although the enhancement in insulin secretion dropped slightly to 1.40fold after 12 h, we still observed this sustained enhancement in insulin secretion over the entire 12 h of our study. 27 ACS Paragon Plus Environment


Figure 5. Sustained enhancement of insulin secretion with AuNP-insDNA uptaken in RIN-5F cells. (a) Insulin secretion profile of RIN-5F cells with time, measured using ELISA, showed significantly more insulin secreted when dosed with AuNP-insDNA compared to baseline secretion level, or RIN-5F dosed with our controls citrate-capped AuNPs, AuNP-p(A)DNA or AuNP-p(T)DNA. (b) Fold change of insulin secretion with respect to baseline at each time point showed that AuNP-insDNA had a sustained enhancement starting from 6 h post-incubation and peak at 10 h with an enhancement


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factor of 1.69-fold. *One-tail t test, p < 0.05, N = 3 (c) Varying dose of AuNP-insDNA at 0 nM, 1 nM, 3 nM and 5 nM at 8 h post-incubation showed a minimal dose of 5 nM AuNP-insDNA was needed for significant enhancement to be observed. *One-tail t test, p < 0.05, N = 3

This strong enhancement in insulin secretion was not observed in our non-hybridizing citrate-capped AuNPs and AuNP-p(A)DNA controls or non-insulin specific hybridizing AuNP-p(T)DNA control, whose enhancement factor were only 1.15-fold, 1.19-fold and 1.20-fold respectively after 10 h of incubation. Since both AuNP-DNA controls experienced similar uptake level in RIN-5F cells as AuNP-insDNA, the diminished enhancement was unlikely attributed to their concentration differences in RIN-5F cells, but more likely due to an absence of specific DNA/mRNA hybridization. Although AuNPp(T)DNA is capable of hybridizing to poly(A)-tail of insulin mRNA, it is non-specific and will also hybridize to any poly(A) tail-containing mRNAs, thereby negating any specific enhancement effect brought by the rapid recycling of ribosomes around solely insulin



mRNAs. The importance of DNA/mRNA hybridization for translation enhancement was already demonstrated in our previous studies26-27, and in this case would grant some degree of specificity for targeted mRNA translation enhancement. Nonetheless, it is still interesting to note that all our controls, including the citratecapped AuNPs without any DNA oligomers also exhibited some slight enhancement (one-tail t test, p < 0.1) of ~1.20-fold increase in insulin secretion even without specific DNA/mRNA hybridization. Such non-specific enhancement in mRNA translation was also observed in our previous studies in cell lysate26-27 and we attributed this to the nonspecific adsorption of intracellular proteins in the cytoplasm which facilitated the recruitment of translation factors and ribosomal proteins close to each other to marginally improve the efficiency of translation. In this study, we also noted that the highest translation enhancement factor of 1.69fold observed for insulin mRNA in RIN-5F cells was lower than that achieved in our cellfree HeLa lysate system of 2.18-fold enhancement26. Here, the large population of nontarget mRNAs in living cells could have interfered with hybridization between insDNA and insulin RNA and consequently led to a lower translation enhancement. Such 30 ACS Paragon Plus Environment

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interference effect was more prominent for AuNP-p(T)DNA as it could bind nonspecifically to multiple species of mRNA with a poly(A) tail, thus resulting in only 1.19fold enhancement factor. The lower enhancement factor achieved in RIN-5F cells could also be attributed to a lower concentration of AuNP-insDNA present in the cytoplasm, due to a poor AuNPinsDNA uptake. This amount present in the cells was also less controllable compared to the concentration of AuNP-DNA present in the HeLa lysate. In our present study, we found that a minimal incubation concentration of 5 nM AuNP-insDNA was required for translation enhancement as reducing its concentration below 5 nM to 3 nM and 1 nM at 8 h post-incubation resulted in progressively lower enhancement factor of 1.68-fold, 1.19-fold and 1.12-fold respectively (Figure 5c).

Functionality of Insulin Secreted Apart from secreting more insulin after the uptake of AuNP-insDNA in RIN-5F cells, it was also important that the insulin secreted possesses the right conformation and functionality as a hormone. Using an insulin bioassay kit that directly detects and 31 ACS Paragon Plus Environment


quantifies functional insulin based on its binding to its receptor, we found that AuNPinsDNA resulted in a similar trend of enhanced functional insulin secretion from 6 h to 12 h, although the peak enhancement occurred later at 12 h with an enhancement factor of 1.64-fold instead of 10 h as observed with ELISA measurement (Figure 6a,b). By taking the ratio of functional insulin and total insulin measured by bioassay and ELISA kit respectively, we determined that all insulin secreted with AuNP-insDNA uptaken in cells had ~100 % functionality over the 12 h of incubation and insulin secretion (Figure 6c).


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Figure 6. Functional insulin secretion with AuNP-insDNA uptaken in RIN-5F cells. (a) Functional insulin secretion profile of RIN-5F cells with time, measured using insulin bioassay, showed significantly more functional insulin secreted when dosed with AuNPinsDNA compared to baseline secretion level, or RIN-5F dosed with our controls citratecapped AuNP, AuNP-p(A)DNA or AuNP-p(T)DNA. (b) Fold change of functional insulin secretion with respect to baseline at each time point showed that AuNP-insDNA had a sustained enhancement starting from 6 h post-incubation and peak at 12 h with an enhancement factor of 1.64-fold. *One-tail t test, p < 0.05, N = 3 (c) Almost all (~100%) insulin secreted at different time points when dosed with AuNPs or AuNP-DNAs were fully functional with no significant difference compared to baseline.

On the other hand, the non-specific ~1.2-fold enhancement in mRNA translation previously observed in our controls using ELISA was not observed in our functional bioassay, suggesting that the insulin secreted could be less functional. Apart from the interference from non-target mRNA and the low uptake of AuNP-insDNA in RIN-5F



leading to their low concentration in cells, our results from the functional insulin assay also suggest that the lower level of 1.64-fold insulin enhancement observed in cell compared to 2.18-fold observed in HeLa lysate26 could also be due to the presence of unfolded protein response (UPR) in cells. An increased in insulin synthesis would cause accumulation of unfolded/misfolded insulin in endoplasmic reticulum (ER) causing ER stress and triggering UPR to slow down translation of insulin mRNA, thus ensuring clearance of not properly folded insulin in ER42. However, too much ER stress could also trigger pancreatic beta cell death43 which was fortunately not observed in our study. Nonetheless, AuNP-insDNA was still capable of consistently secreting elevated level of functional insulin from 6 h to 12 h, without causing RIN-5F cell death.

CONCLUSION In this study, we demonstrated colloidal stability and biocompatibility of AuNP-insDNA for bioapplications with RIN-5F cells, specifically in inducing them to enhance their translation of insulin mRNA. The presence of (2.57 ± 0.12) × 105 AuNP-insDNA per cell caused the translation of insulin mRNA to be enhanced by up to 1.69-fold at 10 h post34 ACS Paragon Plus Environment

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incubation, whereas the absence of insDNA oligomers or its replacement to p(A)DNA or p(T)DNA negated the significant enhancement effect. Using this technique, the insulin secreted were ~100 % functional and could be detected by insulin receptor to give a similar enhancement factor of 1.64-fold at 12 h post-incubation. This study is the first to demonstrate the application of well-designed DNA interface on AuNPs that targets the 3’-UTR region of target mRNA to modulate its translation in cells, with specific application to induce sustained enhanced insulin translation in pancreatic islet cells. With insulin as our protein of interest, we demonstrated a novel use of nanotechnology away from their conventional role as insulin nanocarriers towards one which could potentially modulate the insulin production in a patient’s own pancreatic beta cells as a novel alternative therapeutic approach for treatment of type 2 diabetes mellitus while exploiting the “undesirable” protein corona to do so. The same concept could also be applied to treat other diseases associated with deficient protein synthesis in cells.

AUTHOR INFORMATION 35 ACS Paragon Plus Environment


ORCID Kian Ping Chan: 0000-0003-2202-9608 James Chen Yong Kah: 0000-0002-2247-6929

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

The funding used to support the research of the manuscript was from the Ministry of Education (MOE) AcRF Tier 1 Grant (R-397-000-148-133) and the Agency for Science,


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Technology and Research (A*STAR), Singapore. KP Chan would like to acknowledge the scholarship support from A*STAR Graduate Scholarship.

ABBREVIATIONS PEG, polyethylene glycol (PEG); AuNPs, gold nanoparticles; AuNP-insDNA, insDNA oligomers functionalized on AuNPs; TEM, transmission electron microscopy; DH, hydrodynamic diameter; ζ, zeta potential; FBS, fetal bovine serum; PSH, PenicillinStreptomycin-Glutamine; PBS, phosphate buffered saline; ICP-MS, inductively coupled plasma mass spectrometry; DAPI, 4′,6-diamidino-2- phenylindole dihydrochloride; DLS, dynamic light scattering; ELISA, enzyme-linked immunosorbent assay; UPR, unfolded protein response; ER, endoplasmic reticulum.

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Sustained enhancement of insulin mRNA translation using DNA-functionalized gold nanoparticles (AuNPs) in RIN-5F cells. DNA oligomers specific to 3’untranslated region of insulin mRNA were conjugated on AuNPs (AuNP-insDNA) and uptaken inside RIN5F cell to induce enhanced insulin secretion. A gradual increase in amount of secreted insulin was observed for RIN-5F cell doped with AuNP-insDNA compared to RIN-5F cell alone. The highest 1.64-fold change in insulin secretion elicited by AuNP-insDNA with



respect to baseline occurred at 12 h after incubation. All insulin secreted by RIN-5F cells dosed with AuNP-insDNA was also 100% functional and able to bind to its insulin receptor.


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Figure 1. Schematic showing the application of AuNP-insDNA to enhance insulin production in pancreatic islet RIN-5F cells. (i) AuNP-DNA remained colloidally stable in extracellular environment of RIN-5F cells, while citrate-capped AuNPs aggregated. The stable AuNP-DNA were (ii) taken up into cells and (iii) translation of insulin mRNA was enhanced through recruitment of insulin mRNA and other translation proteins to create a high focal concentration of these translation machineries on AuNP-insDNA. (iv) The insulin produced at enhanced levels remained functional and capable of binding to its receptor. 254x190mm (96 x 96 DPI)

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Figure 2. Characterization of synthesized citrate-capped AuNPs and three types of DNA conjugates; AuNPp(A)DNA, AuNP-p(T)DNA and AuNP-insDNA used in this study. (a) TEM image of synthesized citrate-capped AuNPs and (b) histogram of its size distribution. (c) UV-Vis spectrum of AuNPs showed a defined peak at 519 nm with a red-shift in peak upon DNA conjugation. (d) Hydrodynamic diameter (DH) distribution of AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA determined by dynamic light scattering (DLS) indicated a small increment in DH upon DNA conjugation without forming large aggregates. (e) Zeta potential, ζ, was negative for both AuNPs and AuNP-DNA due to coating of negatively-charged citrate and DNA respectively. (f) All AuNP-DNAs had approximately 80 DNA strands per AuNP after conjugation. 254x190mm (96 x 96 DPI)


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Figure 3. Stability of citrate-capped AuNPs and AuNP-DNA in the RIN-5F extracellular environment. (a) UVVis spectrum showed progressively peak broadening of citrate-capped AuNPs over time indicating gradual aggregation of the AuNPs. On the other hand, a distinct UV-Vis peak with no significant peak broadening was maintained over time for (b) AuNP-p(A)DNA, (c) AuNP-p(T)DNA and (d) AuNP-insDNA. 254x190mm (96 x 96 DPI)



Figure 4. Cellular uptake and toxicity of AuNPs, AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA. (a) Citrate-capped AuNPs showed a progressive increased in their amount in RIN-5F cells with incubation time while a non-increasing amount of AuNP-p(A)DNA, AuNP-p(T)DNA and AuNP-insDNA was observed from 4 h to 12 h. (b) Microscopy images of RIN-5F cells alone and dosed with AuNPs or AuNP-DNA for 6 h were captured. AuNPs were visualized under dark field imaging as bright spots due to their strong optical scattering. They were found to surround cell’s nucleus, which was stained with DAPI. All scale bars are 50 µm. (c) 100% cell viability was observed when RIN-5F cells were incubated with AuNPs or AuNP-DNA for 24 h. 254x190mm (96 x 96 DPI)


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Figure 5. Sustained enhancement of insulin secretion with AuNP-insDNA uptaken in RIN-5F cells. (a) Insulin secretion profile of RIN-5F cells with time, measured using ELISA, showed significantly more insulin secreted when dosed with AuNP-insDNA compared to baseline secretion level, or RIN-5F dosed with our controls citrate-capped AuNPs, AuNP-p(A)DNA or AuNP-p(T)DNA. (b) Fold change of insulin secretion with respect to baseline at each time point showed that AuNP-insDNA had a sustained enhancement starting from 6 h postincubation and peak at 10 h with an enhancement factor of 1.69-fold. *One-tail t test, p < 0.05, N = 3 (c) Varying dose of AuNP-insDNA at 0 nM, 1 nM, 3 nM and 5 nM at 8 h post-incubation showed a minimal dose of 5 nM AuNP-insDNA was needed for significant enhancement to be observed. *One-tail t test, p < 0.05, N =3 254x190mm (96 x 96 DPI)



Figure 6. Functional insulin secretion with AuNP-insDNA uptaken in RIN-5F cells. (a) Functional insulin secretion profile of RIN-5F cells with time, measured using insulin bioassay, showed significantly more functional insulin secreted when dosed with AuNP-insDNA compared to baseline secretion level, or RIN-5F dosed with our controls citrate-capped AuNP, AuNP-p(A)DNA or AuNP-p(T)DNA. (b) Fold change of functional insulin secretion with respect to baseline at each time point showed that AuNP-insDNA had a sustained enhancement starting from 6 h post-incubation and peak at 12 h with an enhancement factor of 1.64-fold. *One-tail t test, p < 0.05, N = 3 (c) Almost all (~100%) insulin secreted at different time points when dosed with AuNPs or AuNP-DNAs were fully functional with no significant difference compared to baseline. 254x190mm (96 x 96 DPI)


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Sustained enhancement of insulin mRNA translation using DNA-functionalized gold nanoparticles (AuNPs) in RIN-5F cells. DNA oligomers specific to 3’untranslated region of insulin mRNA were conjugated on AuNPs (AuNP-insDNA) and uptaken inside RIN-5F cell to induce enhanced insulin secretion. A gradual increase in amount of secreted insulin was observed for RIN-5F cell doped with AuNP-insDNA compared to RIN-5F cell alone. The highest 1.64-fold change in insulin secretion elicited by AuNP-insDNA with respect to baseline occurred at 12 h after incubation. All insulin secreted by RIN-5F cells dosed with AuNP-insDNA was also 100% functional and able to bind to its insulin receptor. 254x190mm (96 x 96 DPI)


Enhanced Secretion of Functional Insulin with DNA-Functionalized

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