PolyMetformin combines carrier and anticancer activities for in vivo siRNA delivery

Metformin, a widely implemented anti-diabetic drug, exhibits potent anticancer efficacies. Herein a polymeric construction of Metformin, PolyMetformin (PolyMet) is successfully synthesized through conjugation of linear polyethylenimine (PEI) with dicyandiamide. The delocalization of cationic charges in the biguanide groups of PolyMet reduces the toxicity of PEI both in vitro and in vivo. Furthermore, the polycationic properties of PolyMet permits capture of siRNA into a core-membrane structured lipid-polycation-hyaluronic acid (LPH) nanoparticle for systemic gene delivery. Advances herein permit LPH-PolyMet nanoparticles to facilitate VEGF siRNA delivery for VEGF knockdown in a human lung cancer xenograft, leading to enhanced tumour suppressive efficacy. Even in the absence of RNAi, LPH-PolyMet nanoparticles act similarly to Metformin and induce antitumour efficacy through activation of the AMPK and inhibition of the mTOR. In essence, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy.

T he discovery of RNA interference (RNAi) has since provided a conserved cellular pathway to modulate or inhibit endogenous gene expression through short interfering RNA (siRNA) therapeutics 1,2 . However, naked siRNA is susceptible to degradation by nucleases in blood serum and unable to cross the cell membrane due to its anionic charge 3,4 . Thus, systemic delivery of RNAi therapeutics to cancerous tissues remains a major challenge. To date, in vivo siRNA delivery has been achieved by vector-based delivery systems with cationic or ionizable characteristics, including polycation 5,6 , lipid [7][8][9] and inorganic nanoparticles 10,11 , and RNA modifications/conjugations 12,13 . Among them, delivery using cationic polymers composed of different cationic compositions has become one of the most widely accepted strategies for delivery of siRNA 14,15 . Yet, the functional mechanisms of a polycation's molecular composition on siRNA delivery remain unclear 16 . Recently, materials have been developed incorporating guanidine groups to increase cellular uptake and transfection 17,18 . Herein a polycationic bi-guanidine composed of the anticancer therapeutic, Metformin (dimethyl-biguanide), has been developed for in vivo siRNA delivery. Metformin, one of the most effective drugs against diabetes 19 , is also known as a therapeutic agent against cancers including lung cancer 20,21 , pancreatic cancer 22 , breast cancer 23 , gastric cancer 24,25 and so on. The anticancer efficacy of Metformin can be primarily attributed to the activation of AMP-activated protein kinase (AMPK) 26,27 and inhibition of the mammalian target of rapamycin (mTOR) 28,29 . This cationic small molecular drug shows excellent tolerability and can be dosed at up to 2 g per day due to low toxicity. Moreover, the cationic biguanide composition of Metformin makes its polymeric form a desirable carrier for siRNA delivery. Therefore, it is expected that polymeric Metformin (PolyMet) would serves dual purposes as both a gene carrier and an antitumour therapeutic to achieve combinational therapeutic efficacies against cancer. Human non-small-cell lung cancer (NSCLC) is a well-known, aggressive and metastatic refractory tumour that responds to treatment by Metformin 30,31 . Thus, a NSCLC tumour cell H460 was used to evaluate the efficacy of siRNA delivery and antitumour abilities of PolyMet in this study.
Our data show a possibility to facilitate vascular endothelial growth factor (VEGF) siRNA in vivo delivery by PolyMet nanoparticles and enhanced tumour growth. In the absence of RNAi, LPH-PolyMet nanoparticles, like metformin, activated the AMPK, inhibited the mTOR pathway and induced tumour autophagy and apoptosis. Thus, PolyMet successfully combines the intrinsic anticancer efficacy of Metformin with the capacity to carry siRNA to enhance the therapeutic activity of an anticancer gene therapy.

Results
Synthesis and characterization of PolyMet polymer. Inspired by the fact that Metformin can be synthesized through a one-step reaction of dimethylamine hydrochloride and 2-cyanoguanidine (dicyandiamide), with heating ( Supplementary Fig. 1), the Metformin polymer was designed using a similar method. To yield PolyMet, linear polyethylenimine (PEI) and dicyandiamide were reacted under heat in acidic conditions (Fig. 1a). Linear PEI hydrochloride (0.2 g) and dicyandiamide (2 g) were mixed in 10 ml 2 M HCl solution. The reaction mixture was reacted at 100°C for 24 h, purified through an ultrafiltration tube to remove excess dicyandiamide, washed with deionized water for two times and lyophilized. The formation of PolyMet was verified by using proton nuclear magnetic resonance ( 1 H-NMR) (Supplementary Fig. 2A) and matrix assisted laser desorption/ ionization time-of-flight (MALDI-TOF) ( Supplementary  Fig. 2B,C) analyses. 1 H-NMR (400 MHz, d6-dimethylsulfoxide) spectrum exhibits all characteristic proton resonance peaks corresponding to the present PolyMet molecules. The appearance of the proton resonance at d 2.86-3.75 p.p.m. in the product along with the characteristic guanidium proton at d 5.80-6.40 p.p.m. confirmed the formation of biguanide polymer. The spectrum also indicated near 95% substitution ratio of PolyMet by comparing the area ratios of PEI (2.53-2.70 p.p.m.) and PolyMet (2.86-3.75 p.p.m.). MALDI-TOF of the PolyMet and free PEI was performed to determine the synthesis of PolyMet ( Supplementary Fig. 2B,C). The distribution centre for PEI (ca. 570 Da) was completely red-shifted compared with PolyMet (ca. 1600 Da), which is consistent with the 1 H-NMR result, indicating successful conjugation of PEI with 2-cyanoguanidine ( Supplementary Fig. 2B). The average molecular weight of PEI shown by MALDI-TOF analysis is smaller than the molecular weight we used for synthesis (ca. molecular weight is 4,300 Da), which might be due to the existence of numerous differently charged species of PEI, since MALDI-TOF only detects singly charged species 32 . Moreover, the extended MALDI-TOF mass spectra ( Supplementary Fig. 2C) show several series of oligomer ions 43 and 129 m/z apart from each other, in agreement with the PEI (43 m/z for C 2 H 5 N unimer) and PolyMet (129 m/z for C 4 H 11 N 5 unimer) repeat units, respectively. As described in authoritative literatures 33,34 , Metformin can be characterized by (1) using ultraviolet absorption at the wavelength of maximum absorbance at about 233 nm or (2) exhibiting a red colour in the solution of nitroprusside/potassium hexacyanoferrate(III)/ sodium hydroxide. Both PolyMet and Metformin exhibited a maximum absorbance around 233 nm (Fig. 1b) and showed red colour in the colour test in a dose-dependent manner (Fig. 1c). These results suggest that after the reaction with dicyandiamide, the secondary amines in PEI had been completely replaced with biguanides.
Notably, PolyMet showed a significantly lower cytotoxicity (Fig. 1d) and threefold higher maximum tolerable dose compared with PEI (Supplementary Table 1), indicating PolyMet containing guanidine is more tolerable than its secondary amine-containing counterpart. But the lower maximum tolerable dose of PolyMet than Metformin (Supplementary Table 1) is perhaps attributed to the interaction of the cationic polymer with the serum proteins in the blood. In summary, PolyMet as a cationic polymer may be used to condense and deliver negatively charged siRNA with similar pharmacological properties and activity to Metformin due to the biguanide structure.
Preparation and characterization of LPH nanoparticles. It is well documented that siRNA forms a loose and unstable complex when mixed with only polycationic polymers due to its low molecular weight and low charge density 35,36 . Therefore, liposomes were used to coat complexes to protect siRNA from dissociation or clearance before it reaches the tumour in our study. First of all, hyaluronic acid (HA), a polyanionic and multivalently charged polysaccharide, was used to facilitate siRNA delivery and enhance the particle condensation. Then cationic 1,2-dioleoyl-3-trimethylammonium-propane chloride salt (DOTAP) liposome was applied to coat complex to prove the stability of complex. These core/shell-type nanoparticles, referred to the lipid-polycation-hyaluronic acid (LPH) nanoparticle were established for systemic delivery of siRNAs to the tumour site 37,38 . Specifically, HA was mixed with siRNA at 1:1 mole ratio, and modulated the N/P ratios of PolyMet/(HA þ siRNA) complex. Aggregates were observed at an N/P ratio around 0.9, where a neutral complex was formed. In this case, 0.6 was chosen as the optimal ratio, as the complex remained negatively charged (B À 20 mV) with a relatively small size (B100 nm). At this ratio the PEI/(HA þ siRNA) complex had a similar charge and size with PolyMet/(HA þ siRNA) complex ( Supplementary Fig. 3). Next, DOTAP/cholesterol (1:1 mol/mol) cationic liposomes were added to the complex to form the lipid coating through charge-charge interactions. Studies have shown that the NSCLC cell H460 overexpresses sigma receptors 17,39,40 , thus a targeting ligand for the sigma receptors, DSPE-PEG-anisamide, was used to deliver nanoparticles to the tumour by the post-insertion method to form the outer leaflet of the liposome membrane. The size, distribution, morphology and the characteristics of the nanoparticles are summarized in Fig. 2 Table 2).

LPH-PolyMet combines siRNA carrier and anticancer activities.
Previous studies have shown that VEGF siRNA suppresses angiogenesis and may be a potential therapeutic agent for NSCLC 41 . Human NSCLC H460 tumour-bearing mice received intravenous (i.v.) injections of different formulations with VEGF siRNA or control siRNA every other day. On successive i.v. injections into tumour-bearing mice, the greatest enhancement of antitumour efficacy was observed when LPH-PolyMet-siVEGF was administered (Fig. 3a).
Notably, the LPH-PolyMet-siCtrl group that contained control siRNA also showed a partial tumour suppression activity, indicating that poly-biguanide structure of PolyMet has the antitumour activity. Meanwhile, minimal suppression of tumour growth was found in the LPH-PEI-siCtrl group. One day after the second injection, mice were killed and tumour lysates were prepared for western blot. As shown in Fig. 3b, LPH-PolyMet-siVEGF group showed robust knockdown of VEGF with about 95% of silencing compared with the phosphate-buffered saline (PBS) group. LPH-PEI-siVEGF had only a partial effect on the VEGF expression level with about 70% of knockdown in comparison with control group. LPH-PolyMet-siCtrl showed a slight knockdown effect. LPH-PEI-siCtrl had no measurable effect on the protein expression level of VEGF.
Terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay further confirmed the induction of apoptotic cells in tumours (Fig. 3c). The number of TUNEL-positive apoptotic cells after LPH-PolyMet-siVEGF treatment was about 40%, which was the highest among all groups. This indicates that elevated apoptosis occurred after downregulation of VEGF expression, which is consistent with studies reported elsewhere 42 . Treatment with LPH-PEI-siVEGF could only induced apoptosis in about 20% of cells. Importantly, we also observed that LPH-PolyMet-siCtrl exhibited significant higher TUNEL-positive population compared with PBS or LPH-PEI-siCtrl, indicating the apoptotic capability of PolyMet gene carrier itself.
PolyMet LPH was then investigated to determine its ability to carry other therapeutic siRNA. As an anti-apoptotic protein BCL-2 promotes cell survival, and inhibition of BCL-2 can suppress tumour growth 43,44 . Herein siRNA used against BCL2 inhibited tumour growth, especially in the LPH-PolyMet-siBCL2 treatment group, which provided persistent suppression of tumour growth ( Supplementary Fig. 4A). The level of BCL-2 after treatment was detected by western blot analysis, LPH-PolyMet-siBCL2 induced a significant downregulation of BCL-2 level in comparison with all other groups ( Supplementary Fig. 4B) and the TUNEL assay further confirmed the induction of apoptotic cells in tumours. The number of TUNEL-positive apoptotic cells after LPH-PolyMet-siBCL2 treatment was about 70%, which was significantly higher than all other groups  Supplementary Fig. 4C). This indicates that elevated apoptosis occurred after downregulation of BCL2 anti-apoptotic protein expression in the LPH-PolyMet-siBCL2 group. PolyMet was then tested for gene delivery and tumour inhibition in a melanoma model. As shown in Supplementary  Fig. 5, the most effective treatment, LPH-PolyMet-siVEGF resulted in a knockdown of VEGF level by B70% and a suppression of tumour growth by about 80% in comparison with the PBS group in a 1205Lu melanoma model. Meanwhile, LPH-PEI-siVEGF also suppressed tumour growth effectively, but not as persistently as LPH-PolyMet-siVEGF.
Moreover, the antitumour activity of PolyMet was compared with other two commonly used polycations, poly-L-lysine (PLL) and protamine. PLL and Protamine were formulated in the LPH systems containing the same amount of VEGF siRNA as PolyMet. The antitumour efficacy and VEGF knockdown were presented in Supplementary Fig. 6. All LPH formulations had significantly suppressed tumour growth in comparison with PBS. However, there was no significant difference among PEI, PLL and protamine. The greatest enhancement of antitumour effect was observed with PolyMet. The expression of VEGF was also evaluated after day 16, as shown in Supplementary Fig. 6B. The VEGF level in the tumour was significantly lower than other groups, as well as the PBS control. Thus, PolyMet showed the greatest antitumour activity among all four polycations tested.

Mechanisms of PolyMet on the inhibition of tumour growth.
Both clinical and preclinical studies have shown that Metformin improved survival and/or antitumour efficacy 25,45 . Results shown in Fig. 3 and Supplementary Figs 4, 5 and 6B indicate the tumour growth inhibition of LPH-PolyMet-siCtrl. It was thus hypothesized that in the absence of RNAi, PolyMet, like its unimer Metformin, may be responsible for some part of anticancer activity. The effect of LPH-PolyMet and different nanoparticles on an H460 xenograft was studied. As shown in Fig. 4, treatment with LPH-PolyMet led to significant inhibition of cancer growth in comparison with PBS. A noticeable difference in tumour growth inhibition was observed between LPH-PEI and LPH-PolyMet (Fig. 4a), suggesting that PolyMet played an important role in enhancing antitumour activity. At the end of the experiment, the tumours of the animals treated with LPH-PolyMet were o1% of the total body weight, which was significantly smaller than the LPH-PEI (4%) and PBS (6%) groups (Fig. 4b). Moreover, no toxicity was detected in blood haematology serum chemistry or the histology of the major tissues ( Supplementary Fig. 7 and Supplementary Table 3).
Furthermore, we compared the effect of LPH-PolyMet siRNA nanoparticles with Metformin in an H460 xenograft ( Supplementary Fig. 8). Free Metformin (1.25 mg kg À 1 ) equivalent to the unimer dose of PolyMet (1.25 mg kg À 1 ) that was required to deliver 0.625 mg kg À 1 of VEGF siRNA was used in the study. It was confirmed that there is no significant difference between the PolyMet carrier (LPH-PolyMet-siCtrl) and Metformin, indicating that biguanide in the PolyMet copolymers are associated with the anticancer activities of Metformin. Both of the groups exhibit significantly higher antitumour growth activity compared with LPH-PEI-siCtrl or PBS while LPH-PolyMet-siVEGF has the best antitumour efficacy. In this case tumour growth was suppressed seven-to eightfold compared with the PBS group, and about twofold compared with the PolyMet carrier or the Metformin group, which suggests again that PolyMet successfully combine the intrinsic anticancer efficacy of Metformin with the tumour suppressive effects of VEGF siRNA to enhance the therapeutic activity of an anticancer gene therapy.
It has been reported that Metformin inhibits cancer cells by activating the AMPK and inhibiting the mTOR pathways 28,46 . We checked the activity of PolyMet in these pathways and compared it with that of Metformin. AMPK acts as a metabolic tumour suppressor that governs glucose and lipid metabolism 47 . In many cases, a low level of phosphorylation of AMPK is correlated with poor prognosis after cancer treatment 48 .
We observed that following treatment with Metformin or LPH-PolyMet, phosphorylation levels of AMPKa in the H460 tumour were significantly enhanced compared with the PBS group (Fig. 5a). The activation of AMPKa did not occur in the LPH-PEI group, suggesting PolyMet is the key factor in the nanoparticle that can activate AMPKa pathway. mTOR is a downstream effector of AMPK 49 . AMPK activation inhibits mTOR and its downstream effector kinases 50 . Phosphorylation of mTOR plays a pivotal role in the proliferation and survival of cancer cells 51 . Therefore, we then evaluated the effects of treatments on the activity of mTOR (Fig. 5a). Metformin and LPH-PolyMet treatments led to a significant inhibition of mTOR activity indicated by stronger reduction in p-mTOR/mTOR levels compared with the PBS group. The activity was not shared by LPH-PEI. Autophagy is recognized as a potentially toxic mechanism for Metformin that results in the inhibition of cancer growth 52,53 . We evaluated whether autophagy can also be observed after treatment of LPH-PolyMet. Microtubule-associated protein light chain 3 b (LC3b) is a specific marker for autophagy initiation 54 . As shown  Fig. 5b, Metformin-and LPH-PolyMet-treated tumours showed a higher LC3b-associated red fluorescence than other groups, indicating that both Metformin and LPH-PolyMet could induce autophagy in the H460 lung cancer xenograft model. Finally, the mechanism of the antitumour efficacy after treatment was determined by the TUNEL assay, a common method to test for apoptosis (Fig. 5b). The per cent of apoptotic cells after Metformin or LPH-PolyMet treatments was 12.4% and 15%, respectively, while no significant apoptosis induction was observed in other groups. This suggests that Metformin or polymer form can induce cell apoptosis and play a critical role in regulating the cancer cell survival.

Discussion
Nanotechnology has been extensively exploited to prolong the circulation time and to enhance the therapeutic activity for a variety of drugs. After systemic injection the nanoparticles drain through the leaky tumour blood vessels and are retained at the tumour site, resulting in increased tumour accumulation of the nanoparticle-formulated drug compared with the free drug. This well-documented phenomenon is referred to as the enhanced permeability and retention effect 55 . Thus, nanoparticles offer a number of advantages over free drugs, which make them an ideal delivery vehicle for Metformin. However, due to its high hydrophilicity, Metformin cannot be effectively loaded into established nanocarriers, which explains why only few Metformin nanoparticles have been reported [56][57][58] . Recently, Yang et al. 56 reported the co-encapsulation of epirubicin and Metformin into liposomes through remote loading. This nanoparticle successfully inhibited the growth of CD133 þ cancer stem-like cells. Jose et al. 57 developed Metformin-loaded bovine serum albumin nanoparticles (MET-BSA) by the coacervation method and observed that MET-BSA nanoparticle showed higher efficacy than Metformin alone or the BSA carrier itself. However, PolyMet, which combines both gene delivery and anticancer capabilities, represents a novel approach.
Guanidination of a polymer enhances the activity for gene delivery and has considerably less toxicity compared with primary amines of the polymer 59,60 . The guanidine has been found to readily pass through the non-polar membrane of a cell and even across tissue barriers by possibly forming a bidentate hydrogen bond with anionic cell surface phosphate, carboxylates and/or sulfates on the cell surface 61,62 . Materials have been previously developed incorporating guanidine groups to increase cellular uptake and transfection 60,[63][64][65] . For example, Lee et al. 60 used pyrazole-1-carboxamidine to react with branched PEI, and observed enhancement of transfection efficiency and decreased cytotoxicity of PEI by guanidylation. Wender et al. developed non-natural guanidinium-rich molecular transporters to enhance cellular uptake 61,66 , through tunable polyguanidino dendrimers, and observed higher cellular uptake 65 . Guanidinium-rich molecular transporters can penetrate cells and overcome efflux-mediated multidrug resistance 67 . Although these previously reported polyguanidines are established gene carriers, the carriers themselves fail to exhibit anticancer activity without their therapeutic agent. Thus, PolyMet serves as both a gene carrier and anticancer therapeutic. However, findings presented herein have not been investigated with PolyMet against a p53-deficient tumour, which remains to be further explored. The focus of this study is to show the carrier and anticancer activities of PolyMet. The dose of PolyMet used in the study was dictated by the dose of siRNA and was only a fraction of the effective dose of metformin. Further study with increased PolyMet dose will bring the new polymer a step closer to the clinical translation. Cell culture. H460 human NSCLC cells and 1205Lu melanoma cancer cells were originally obtained from American Type Culture Collection and were cultured in DMEM medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Life Technologies, Carlsbad, CA), 100 U ml À 1 penicillin and 100 mg ml À 1 streptomycin (Invitrogen). Cells were cultured in a humidified incubator at 37°C and 5% CO 2 .
Experimental animals. Female nude female mice and female CD1 mice that were 6-8 weeks old were used in the studies. glucose solution A (containing the 12.5 mg of siRNA and 12.5 mg of HA) with 100 ml of 5% glucose solution B (containing 5 mg of PEI, or 25 mg of PolyMet, or 6 mg of PLL, or 13 mg of Protamine) and incubated at room temperature for 10 min. The LPH cores were then mixed with 30 ml of cationic DOTAP/cholesterol liposomes and incubated at room temperature for 10 min to ensure proper lipid coating. The lipid-coated nanoparticles were PEGylated using a post-insertional approach by adding 10 ml DSPE-PEG (10 mg ml À 1 ) and 10 ml DSPE-PEG-anisamide (10 mg ml À 1 ) and incubating the nanoparticles at 50°C for 15 min.
Characterization of LPH NPs. The size and surface charge of LPH cores and final particles were determined by using a Malvern ZS90 Zetasizer (Worcestershire, UK). Transmission electron microscope images of LPH were acquired through the use of JEOL 100CX II transmission electron microscope (Tokyo, Japan). Briefly, freshly prepared LPH nanoparticles (5 ml) were carefully dropped onto a 300-mesh carbon-coated copper grid (Ted Pella, Inc., Redding, CA) and allowed to stand at room temperature for 5 min. Grids were then stained with 1% uranyl acetate (5 ml) and allowed to incubate briefly (10 s) and quickly dry. All images were acquired at an accelerating voltage of 100 kV. Western blot analysis. Western blotting was used to evaluate the protein expression of tumour cells after systemic administration. Tumour-bearing mice were injected i.v. every other day and were killed 24 h after the second injection. A unit of 50 mg of tumour was collected randomly via biopsy and homogenized with a Tissue Master-125 homogenizer (OMNI International) in 500 ml cold radioimmunoprecipitation assay buffer (Sigma-Aldrich) supplemented with 1% protease inhibitor cocktail (Sigma-Aldrich) for 30 min. Total homogenate was then spun down at 13,000g with a tabletop centrifuge for 10 min at 4°C. Supernatant was collected and protein sample was quantified using a bicinchoninic acid assay kit in accordance with the manufacturer's instruction (Pierce Biotech). Samples were diluted in 4 Â sample buffer and 10 Â sample-reducing reagent (Thermo Fisher). A unit of 40 mg protein from each group was separated by 4-12% SDS-PAGE electrophoresis (Invitrogen) and transferred to a polyvinylidene difluoride membrane (Bio-Rad). Membranes were blocked with 5% non-fat dry milk (Bio-Rad) for 1 h at room temperature and then incubated with primary antibodies (1:1,000 dilution; Cell Signal) overnight at 4°C. The membranes were washed three times with PBS-Tween, followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:4,000 dilution; Cell Signal) for 1 h at room temperature. Finally, the membranes were washed three times with PBS-Tween and developed using Pierce ECL Western Blotting substrate (Thermo Scientific). The expression level of targeted protein was quantified by ImageJ software (NIH). GAPDH was detected as loading control. Images have been cropped for presentation but are otherwise unmodified. Full-size images are presented in Supplementary Figs 9 and 10.
Immunostaining. The TUNEL assay was used to detect apoptotic tumour cells after systemic administration. Briefly, tumours were collected and fixed in freshly prepared 4% paraformaldehyde or 10% formalin for 1 day. Tumour samples were paraffin-embedded and sectioned by the UNC Animal Histopathology Core. The slides were then deparaffinized in xylene and rehydrated through a gradient alcohol series. The TUNEL staining was performed in accordance with the manufacturer's protocol (Promega). Nuclei of tumour cells were stained with DAPI (Vector Laboratories). Images of were taken using a fluorescence microscope (Nikon). The percentage of apoptotic cells was calculated by dividing the number of apoptotic cells from the total number of cells in each microscopic field. Representative microscopic fields (n ¼ 5) were randomly selected in each treatment group for this analysis.
Statistics analysis. All statistical analyses were carried out using GraphPad Prism Software (Version 5.0, GrapPad Software, San Diego, CA).