Novel Methods for Delivering and Promoting the Endosomal Escape of Nucleic Acid Based Drugs: Chiral Polyamines and Hydrophobic Nanoparticle-Containing Liposomes

Nucleic acid based drugs such as plasmid DNA (pDNA), small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), and both antisense and antigene oligonucleotides, are potentially potent and specific compounds for therapeutic applications. Many major life threatening ailments might be treated using these molecules, and many polynucleotide products are currently in advanced clinical development. However, their successful therapeutic application is hindered due to limited delivery to their site of action in either the cytosol or the nucleus of a cell. Some of the barriers in the path of successful delivery of these biomolecules to their site of action have been addressed. However, endosomal entrapment followed by maturation to lysosomes and degradation of these compounds inside the cell is one remaining major hurdle. This dissertation describes two novel siRNA delivery techniques which present distinct advantages in their respective areas of application while, at the same time, constitute promising platforms for developing therapeutic biologicals. Chapter 2 focuses on liposomal delivery vehicles containing hydrophobic nanoparticles in their bilayers, which encapsulate the nucleic acid based drugs and promote endosomal escape by nanoparticle induced fusion with the endosomal membranes. Specifically we use metal nanoparticles in specialized liposomes for the efficient delivery of small interfering RNA (siRNA). Manuscript 2 focuses on novel chiral cationic polymers – polyethyleneimines (PEIs) – that form complexes with the negatively charged nucleic-acid based drugs and promote endosomal escape via a proton sponge effect. Specifically we use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalentlylinked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. We found that two of the designed polymers improved transfection efficiency relative to commercially available transfection reagents with lower cell toxicity. In total this dissertation presents work that demonstrates novel and efficient delivery strategies that promote endosomal escape and enhance the intracellular activity of nucleic acid based drugs.

development. However, their successful therapeutic application is hindered due to limited delivery to their site of action in either the cytosol or the nucleus of a cell. Some of the barriers in the path of successful delivery of these biomolecules to their site of action have been addressed. However, endosomal entrapment followed by maturation to lysosomes and degradation of these compounds inside the cell is one remaining major hurdle. This dissertation describes two novel siRNA delivery techniques which present distinct advantages in their respective areas of application while, at the same time, constitute promising platforms for developing therapeutic biologicals. Chapter 2 focuses on liposomal delivery vehicles containing hydrophobic nanoparticles in their bilayers, which encapsulate the nucleic acid based drugs and promote endosomal escape by nanoparticle induced fusion with the endosomal membranes. Specifically we use metal nanoparticles in specialized liposomes for the efficient delivery of small interfering RNA (siRNA). Manuscript 2 focuses on novel chiral cationic polymers -polyethyleneimines (PEIs) -that form complexes with the negatively charged nucleic-acid based drugs and promote endosomal escape via a proton sponge effect. Specifically we use of chiral cationic polyamines for two intriguing applications: fabrication of chiral covalently-linked microcapsules, and enantiospecific delivery of siRNA to Huh 7 cells. We found that two of the designed polymers improved transfection efficiency relative to commercially available transfection reagents with lower cell toxicity. In total this dissertation presents work that demonstrates novel and efficient delivery strategies that promote endosomal escape and enhance the intracellular activity of nucleic acid based drugs. Dr. King, not only is fair and wise but is also realistic and focused in bringing out the best in me under such extreme conditions, so that I perform to my best ability. All these following deadlines were only possible with Dr. King's undaunting support and mentoring of me through out the process.
I also want to Thank Dr. David Rowley and Dr. David Worthen, who have been standing rock solid to support students like me with extreme care and sensitivity. Dr.
Rowley is truly dynamic and quick with his honest support for students and does his best as the Department chair of BPS. I am very Thankful to him for his strong support in my v journey for completing this PhD. I cannot find enough words of acknowledgment and my gratefulness for having Dr. David Worthen both in my PhD committee as well as one of the major co-advisor on the project we did together. Dr. Worthen always has the best interest for his students and is extremely approachable to them at every time of need. I am truly blessed to receive his mentorship and guidance at every step of my program. He has not only taught me scientific and critical reasoning, but also walked with me on the path of all the challenging times, that I faced through out my journey in URI. He exhibits a very strong character and works effortlessly around the clock for all his students. Dr.
Worthen's strong scientific knowledge along with his patient and focused attitude has tremendously brought out the best results through the project we have done together. He is undoubtedly, the best mentor I could ask for and completes my circle of having a "Guru" in my life. I really thank him for everything that he has helped me with. I wish the best for him. The Ombudmun team, Prof Gerry Tyler and Prof. Alfred Killilea provided me with a staunch support at every step and gave me the right emotional and moral support to survive the sensitive, challenging situations at URI. They are extremely precious and like "a feather in my cap" in pursuit of the hard-earned Doctorate degree. I must not forget to Thank Dean Rusnock, whose absolute and wonderful humbleness has provided me with significant emotional/work balance while she considered me for another opportunity to still complete my PhD under the absolutely stressful situations in URI. Her faith in helping students like me is clearly visible and I offer my sincere Thank you for her support, as I prepare myself to walk with honor with a very hard earned degree.
vi I would like to acknowledge the role played by my best friend, colleague, undergraduate classmate and my confidante in life, Dr. Ashish L. Sarode. This is a man who stood strongly by my side through out the entire time, ever since I entered URI, to achieve my dream of completing my Ph.D. Just like any other best friends, we would always talk about our big dreams to work together on our aims one day. We wanted to contribute in areas of Cancer and Vitiligo, since these diseases have directly affected our personal lives in the history of our family. My maternal grandfather fought in World War -II and came back home with an incurable, late stage terminal stomach cancer. Ashish's mother has Vitiligo, which later got passed to his sister. Life came to a full circle when we finally crafted our dream research project, in honor to contribute towards autoimmune diseases, that now stands as a big hit with the major leading Biopharmaceutical   #69} Hence, in order to use these drugs therapeutically, it is necessary to develop vehicles and methods for their efficient delivery to their site of action.
Among various potential delivery approaches, the use of self-assembling lipids in order to develop vesicular delivery vehicles has proven to be one of the successful and feasible approaches. 5 In particular, small unilamellar vesicles (SUVs) such as liposomes was shown using Western blot.

Introduction
Pharmaceutical research, in both academia and in industry, is increasingly focused on the development of biotechnology-derived and genetically-engineered nucleic acid 6 based drugs such as plasmid DNA (pDNA, small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), antisense, and antigene oligonucleotides as potential therapeutics. These molecules are highly specific and potent and can be used to treat various life-threatening ailments. 1 Allied Market Research TM has predicted that the global siRNA therapeutics market alone is expected to reach $1.2 billion by the year 2020. While promising as therapeutic agents, the delivery of these molecules is one of the most important and challenging aspects of their development, and is the focus of extensive industrial and academic research efforts. 2 Nucleic acid-based drugs are very hydrophilic, are of high molecular weight, are often chemically-and enzymatically unstable, and are highly charged molecules. 3 If administered naked, these molecules face many hurdles before they reach their target site of action, such as rapid renal clearance, serum degradation, opsonization, RES uptake and metabolism, insufficient tissue and cell internalization, and endosomal degradation, as well as immunosensitization. 4 Hence, in order to use these drugs therapeutically, it is necessary to develop vehicles for their efficient delivery to their site of action. Among various potential formulation methods, the use of self-assembling lipids and polymers in order to develop vesicular delivery vehicles such as lipsomes and polymersomes has proven to be one of the successful and feasible approaches. Despite many efforts and advances in these delivery vehicles, endosomal degradation of their cargo remains one of the pivotal challenges. Hence, there is a pressing need to develop novel techniques to promote endosomal escape of biologics before they are degraded in the endosome. The present investigation describes a novel method designed to address this need. 7 Liposomal delivery systems are popular carriers for nucleic acid-based drugs because of their favorable characteristics such as biocompatibility, biodegradability, spontaneous self-assembly, the ease of large-scale production, and suitability for clinical application 5 . Some of the barriers in the path of efficient delivery of nucleic acid based drugs to their site of action have been addressed using liposomal delivery systems 6  Endosomal degradation is still one of the major barriers to the efficient delivery of nucleic acid-based drugs 8 . Various approaches, such as fusion in the endosomal membrane, a proton sponge effect, pore formation in the endosomal membrane, and photochemical disruption of the endosomal membrane have been assessed in order to address this issue 9 . From among these strategies, the fusion of the liposomal and 8 endosomal membranes and the subsequent release of the liposomal cargo into the cytosol has been perhaps the most promising one 10 . This membrane fusion occurs via inverted hexagonal (H II ) phase formation between liposomal and endosomal bilayers. The H II phase formation can be enhanced by increasing negative interfacial curvature of the liposomal bilayer using lipids with appropriate critical packing parameters 11 . For instance, by using lipids with higher unsaturation in their chains generates a kink that assists in H II phase formation by increasing negative interfacial curvature [12][13][14] . A packing frustration is generated in the hydrophobic domains of the lipids while the H II phase is being formed due to creation of voids around the hydrophilic channels of the H II phase 15 .
In this investigation we hypothesized that the presence of free flowing hydrophobic

Preparation of liposomes
Liposomes were prepared by thin film hydration method. 16 Briefly, a chloroform solution of cationic lipid DOTMA and non-ionic lipid DSPC at an equal molar ratio with/without hydrophobic NPs at various ratios of lipid molecules : NPs was prepared in a glass vial. A thin uniform film was then prepared by rapidly evaporating the organic solvent under vacuum for 2 hours in order to remove the trace solvent. This film was then hydrated using 0.5 ml phosphate buffer saline (PBS) with/without nucleic acid (pDNA or siRNA) by vortexing for 30 seconds. The resulting dispersion was then sonicated using a bath sonicator for 30 minutes. The total lipid concentration in the liposomes was 2 mM.
The total pDNA concentration was 40 ng/µl and that of siRNA was 400 nM, respectively, in the liposomes containing corresponding nucleic acids.

Preparation of multi lamellar vesicles (MLVs)
MLVs were prepared by a thin film hydration method in order to study potential

Dynamic light scattering (DLS)
A Zetasizer Nano ZS (Malvern Instruments Ltd., Worcestershire, UK) was used to perform DLS experiments in order to measure the size of liposomes. A suspension of each liposome preparation (0.5 ml) was deposed into disposable polystyrene cuvettes (Sarstedt AG & Co., Newton, NC, USA) having a 1 cm path length. The temperature was set to 25 °C for the analysis and samples were allowed to equilibrate for 60 seconds before the measurement. The particle size was then determined at a manual setting of 15 counts with 10 seconds per count and a measurement angle of 173° Backscatter (NIBS Default).

Cryogenic transmission electron microscopy (Cryo-TEM)
Cryo-TEM was performed in order to determine the morphology of the liposomes. The samples were prepared at 37 °C using a Vitrobot (FEI Company), a PC-

Phosphorus-31 nuclear magnetic resonance spectroscopy
The 31 P-NMR spectra were acquired on an Agilent NMRS 500 NMR spectrometer operating at 202.3 MHz using a 5-mm OneNMR probe. NMR data were collected for 60 K scans with a 35.7-kHz sweep width using 131 K data points.
Acquisition time was 1.3 s with a relaxation delay of 0.5 s. The data were processed with Mnova program V8.1 Mesterlab research SL. A line broadening of 50 Hz was applied to all spectra. All spectra were indirectly referenced to H 3 PO 4 set to 0 ppm. Data were acquired without spinning.

Cell transfection
Huh-7 cells at 2x10 5 cells/well concentration were seeded in 12 well plates and transfected in the presence of 1 ml/well OPTIMEM-1 using 50 µl/well liposomes for eGFP expressing pDNA experiments. For mFXRa1 protein expressing pDNA experiments, Huh-7 cells were seeded at 4x10 5 cells/well concentration in 6 well plates and transfected in the presence of 2 ml/well OPTIMEM-1 using 100 µl/well liposomes.
For eGFP specific silencing siRNA experiments, 12 well plate conditions as described above were used and Huh-7 cells were first transfected with eGFP expressing pDNA at 2 µg/well concentration using GenJet. The medium was then replaced after 12 hours in order to remove left-over GenJet reagent and the cells were transfected using 50 µl/well liposomes comprising eGFP specific silencing siRNA.

Fluorescence microscopy
Cells transfected using eGFP expressing pDNA or eGFP silencing siRNA were observed 48 hours after transfection under a fluorescence microscope (Eclipse TE2000-E, Nikon Instruments Inc., NY, USA) at a 10x magnification set up in order to detect the eGFP fluorescence in the cells. Images were taken in order to observe the relative eGFP fluorescence in the cells.

FACS analysis
FACS analysis was performed in order to count eGFP-expressing cells using a flow cytometer (BD FACSVerse TM , BD Biosciences, CA, USA). The well plates were removed from the incubator 48 hours after transfection and the medium was discarded.

13
The cells were then washed twice with 1 ml of PBS equilibrated at 37 °C. The cells were then trypsinized using 1 ml of trypsin equilibrated at 37 °C. DMEM (1 ml) containing 10 % FBS, 1 % NEA, and 1 % PS equilibrated at 37 °C was added into the wells. The plate was then shaken and the contents (total 2 ml) were transferred to 15 ml tubes. The tubes were then centrifuged for 5 minutes at 1000 rpm and 5 °C, supernatant was discarded, and 2 ml of PBS stored at 5 °C was added to these tubes. This step was repeated one more time and then 2 ml of room temperature PBS was added to the tubes. The cells were then suspended by pipetting up and down and then analyzed using the flow cytometer with laser set up for counting eGFP expressing cells.

Western blot
The Western blotting technique was used in order to determine the protein levels of mFXRa1 protein from the Huh7 whole cell lysates. Cells were ground 48 hours after transfection in 1x sucrose-Tris buffer using a mechanical homogenizer. The whole cell lysate was then centrifuged at 15000g for 15 minutes to obtain the clear lysate. The protein concentration was determined using the Micro-BCA method from Thermo-Fischer's Pierce Protein protocol. Thirty µg of protein was loaded onto an SDS PAGE gel followed by a semi-wet transfer of the separated proteins from the gel to a PVDF membrane. The membrane containing proteins were blocked in 5% milk/TBST buffer for 3 hours followed by treatment with the primary antibody of mFXRa1 (1:1000) in 5% milk/TBST overnight at 4 °C. The membrane was then washed and incubated with the secondary antibody (1:4000) in 5% milk for 1 hour at room temperature on a lab shaker.
The membranes were washed three times using 1x Tris-sucrose buffer containing (10%) Triton X-100 (TBST) and were then imaged using the chemi-luminescent substrate from Thermo-Fischer Scientific. Protein expression was quantified using a Typhoon 9000-FLA imager and normalized against GAPDH, as an internal housekeeping gene.

Enhancement in bilayer fusogenicity due to presence of NPs
The The enhancement in fusogenicity was assessed by measuring the H II phase transition temperature and by 31 P-NMR analysis. The morphology of MLVs produced for this measurement has been depicted in Figure 4a. As shown in Figure 4b, these MLVs depicted a characteristic 31 P-NMR profile with a high field peak and a low field shoulder 20 pattern, which transformed into a low field peak and a high field shoulder upon transition to the H II phase. As illustrated in Figures 4b and 4c, the phase transition temperature was reduced a from 50 °C to 40 °C upon incorporating 2 nm AuNPs at 10,000:1 lipid molecules : NPs ratio into the bilayers. Although the phase transition temperature was not further reduced, the intensity of the low field peak was higher for a 5,000:1 lipid molecules : NPs ratio at 40 °C for this system (Figure 4d). Moreover, the phase transition temperature was further reduced to 35 °C upon the incorporation of 4 nm AgNPs at a 10,000:1 lipid molecules : NPs ratio into the bilayers (Figures 4e). These data suggest that the phase transition from bilayer to H II was induced not only by increasing the concentration of the NPs but also by an increase in their size.

Hydrophobic NPs improved transfection efficiency
The expression of eGFP was significantly increased by using liposomes containing AuNPs as compared to those without NPs. As depicted in Figure 5a  After counting these eGFP expressing cells, it was determined that the transfection efficiency of eGFP expressing pDNA was enhanced 8-fold by incorporating 4 nm AgNPs into the pDNA-containing liposomes (Figure 6b) as compared to a 1.28-fold increase by incorporating 2 nm AuNPs (Figure 5b). Thus, liposomes containing the larger AgNPs that displayed higher fusogenicity also exhibited higher transfection efficiency as compared to liposomes containing smaller AuNPs.

Conclusions
Upon entering a cell, liposomes containing nucleic acid-based bioactive molecules tend to be taken up by endocytosis, and their therapeutic action depends upon escape from these endosomes into the cytosol. As demonstrated in this work, NPs incorporation into liposomal membranes induced fusogenicity in the liposomal bilayers and led to higher transfection efficiency and biological activity for two major nucleic acid   indicating the polymers' ability to transfect siRNA efficiently (Table 1). More interestingly, compounds R-2a-13 and R-6-13, which are identical except for the threedimensional configuration of the benzyl group, transfect siRNA with approximately the same efficiency as Lipofectamine and Genjet, and substantially lower than the "enantiomeric" polymers.
39 Figure 2 Chart of the intracellular fluorescence of Huh7 cells after transfection with siRNA (all PEIs were used at a 1000 nM final concentration) The chirality of the side chains of the PEIs thus has a direct and measurable effect on the ability of PEIs to transfect siRNA efficiently: S chiral centers (compounds S-6-13 and S-2a-13) transfect siRNA more efficiently than the R analogues. Such a result may seem intuitive: that the interaction of two chiral macromolecules (chiral PEI and chiral siRNA) depends on the three-dimensional configuration of both molecules. This intuition is borne out by the results of this study, which is the first direct proof that the chirality of a polyamine directly impacts its transfection efficiency. Similar effects of the chirality on transfection efficiency were recently observed for the lipid delivery agent 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). 39 In that report, the R enantiomer performed better than either the S enantiomer or the racemic DOTAP mixture.
The toxicity of the newly synthesized PEIs was tested using an MTT assay. of these and other experiments will be reported in due course.

Supplementary Material
Refer  Similarly, upon exposure to the near infra-red and UV radiation, AuNPs demonstrate increase in temperature {An, 2013 #12}. In the proposed future work the hydrophobic NPs such as MNPs and AuNPs will be incorporated into the bilayer of liposomes containing nucleic acid based drugs. These liposomes will be used to transfect the cells.
The cells will then be exposed to the radiofrequency and near infra red/UV radiation for MNPs and AuNPs containing liposomes, respectively. The enhancement in transfection efficiency due to the radiation will be determined by running the same experiment without exposure to the radiation. The cells will be exposed to the radiation post transfection at different starting time points and the optimum time and duration of exposure will be determined. Further, in-vivo experiments will be conducted in mice by dosing them with these specialized liposomes containing nucleic acid based drugs via tail vein injection. The animals will then be exposed to the radiation and the enhancement in protein production with or without radiation exposure will be compared. Fluorescent labeled lipids and nucleic acid based drugs will be used in order to determine the location of the liposomes and exposure to the radiation will be initiated when the fluorescence is Such PEG-lipids with targeting ligand to the distal end of the PEG will be used in our specialized liposomes containing hydrophobic NPs in order to target the liver.
In the third part of the future work, liver targeting approach using our specialized liposomes will be combined with controlled radiation exposure. hypothesize that the hepatocyte targeting liposomes containing NPs will be taken up by the cells in approximately 11 minutes post transfection and if the cells are exposed to the radiation it may lead to endosomal escape and highly efficient transfection of nucleic acid based drugs. Therefore, in-vitro work in intact hepatocytes will be conducted in order to test the proof of concept using specialized liposomes containing targeting moiety and NPs followed by in-vivo work using fluorescently labeled lipids/nucleic acid based drugs.

SUMMARY AND CONCLUSIONS
Despite of the significant potential of nucleic acid based drugs as therapeutic agents for several life threatening ailments, their successful application is majorly limited due to insufficient delivery of these drugs to their site of action. One of the major barriers in the path of their delivery is endosomal degradation, which occurs followed by cellular uptake. Liposomal vehicles such as SNALPs (stable nucleic acid lipid particles) developed by Tekmira is one of the leading strategies for therapeutic application due to its feasibility of large scale and cGMP manufacture, storage stability for up to 2 years, lower in-vivo toxicity, and higher potency and encapsulation efficiency. On the other hand, poly-cationic polymers are majorly used in intact cells for transfection in order to evaluate initial effectiveness of nucleic acid based drugs during discovery stages as well as to understand the etiology of diseases. These poly-cationic polymers are highly efficient in cellular uptake via interaction with negatively charged cell membrane and endosomal escape via proton sponge effect mechanism, however, exhibit higher toxicity.
In this work we demonstrated that the efficiency of SNALP type vehicles was significantly enhanced using hydrophobic NPs, which could be attributed to the ability of these vehicles in efficiently delivering the cargo to the site of action via improved endosomal escape. Whereas, our novel chiral polyamines exhibited higher transfeciton efficiency as well as lower toxicity. Thus, our novel liposomal-and chiral polyamine based vehicles could be beneficial in improving the efficiency of nucleic acid based drugs for therapeutic application and discovery purposes, respectively.
In manuscript 1 we demonstrated that the fusogenicity of liposomal delivery Bile acid homeostasis is achieved through a tightly regulated enterohepatic circulation of bile acids. Canalicular secretion of bile acids through bile salt export pump (BSEP) is the rate-limiting step in such circulation (8,9). Modulation of BSEP expression or function by inherited or acquired factors has a profound impact on the biliary and intrahepatic bile acid levels. Indeed, the impairment of BSEP expression or function has been directly linked to such diseases as progressive familial intrahepatic cholestasis type 2 (10, 11), benign recurrent intrahepatic cholestasis (12,13), and ICP (14,-16).
Under physiological conditions, BSEP expression is coordinately regulated by distinct but related transactivation pathways (17,-21), notably the bile acids/farnesoid X receptor (FXR) signaling pathway (17,18). Activation of FXR by bile acids strongly induces 62 BSEP expression in vitro and in vivo (17,18). Such feed-forward regulation of BSEP by bile acid/FXR is considered a major mechanism for preventing excessive accumulation of toxic bile acids in hepatocytes.
We previously reported that BSEP expression was significantly repressed in the late stages of pregnancy in mice and inversely correlated with serum estrogen 17β-estradiol (E2) levels (22). Further studies showed that E2 repressed BSEP expression in vitro and in vivo through estrogen receptor (ER)-α, and such repression was resulted from a cross talk between the E2/ERα and bile acids/FXR signaling pathway. It is thus concluded that E2-mediated transrepression of BSEP represents an etiological contributing factor to ICP.
However, the underlying mechanisms of such transrepression are not fully understood.
In this study, we demonstrated that E2 repressed BSEP expression through decreasing recruitment of coactivator peroxisome proliferator-activated receptor gamma coactivator- DengR@mail.uri.edu.

Abstract
Bile salt export pump (BSEP) is responsible for biliary secretion of bile acids, a rate