Surface Coating Approach to Overcome Mucosal Entrapment of DNA

Subscriber access provided by BUFFALO STATE

Biological and Medical Applications of Materials and Interfaces

A Surface Coating Approach to Overcome Mucosal Entrapment of DNA Nanoparticles for Oral Gene Delivery of Glucagon-like Peptide 1 Tianqi Nie, Zhiyu He, Yang Zhou, Jinchang Zhu, Kuntao Chen, Lixin Liu, Kam W. Leong, Hai-Quan Mao, and Yongming Chen ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b10294 • Publication Date (Web): 26 Jul 2019 Downloaded from pubs.acs.org on July 28, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 28 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

ACS Applied Materials & Interfaces

A Surface Coating Approach to Overcome Mucosal Entrapment of DNA Nanoparticles for Oral Gene Delivery of Glucagon-like Peptide 1 Tianqi Niea,b,§, Zhiyu Hea,b,c,d,e,§,*, Yang Zhouc,d, Jinchang Zhuc,d, Kuntao Chenc,d, Lixin Liua,b, Kam W. Leonga,b,g, Hai-Quan Maoa,c,d,e,f,*, and Yongming Chena,b,*

aSchool

of Materials Science and Engineering, Sun Yat-sen University, Guangzhou 510275, China

bCenter

for Functional Biomaterials, and Key Laboratory for Polymeric Composite and Functional

Materials of Ministry of Education, Sun Yat-sen University, Guangzhou 510275, China cDepartment

of Materials Science and Engineering, Whiting School of Engineering, Johns Hopkins

University, Baltimore, MD 21218, USA dInstitute

for NanoBioTechnology, Johns Hopkins University, Baltimore, MD 21218, USA

eTranslational

Tissue Engineering Center, Johns Hopkins University School of Medicine, Baltimore,

MD 21287, USA fDepartment

of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore,

MD 21287, USA gDepartment

of Biomedical Engineering, Columbia University, New York, NY 10027, USA

ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 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

Page 2 of 28

ABSTRACT: Oral delivery of nucleic acid therapy is a promising strategy in treating various diseases due to its higher patient compliance and therapeutic efficiency compared to parenteral routes of administration. However, its success has been limited by the low transfection efficiency resulting from nucleic acid entrapment in the mucus layer and epithelial barrier of gastrointestinal (GI) tract. Herein, we describe an approach to overcome this phenomenon and improve oral DNA delivery in the context of treating type II diabetes (T2D). Linear PEI (lPEI) was used as a carrier to form complexes with plasmid DNA encoding glucagon-like peptide 1 (GLP-1), a common target in T2D treatments. These nanoparticles

were

then

coated

with

1,2-dipalmitoyl-sn-glycero-3-phosphocholine

a

mixture

(DPPC)

of and

1,2-dimyristoyl-rac-glycero-3-methoxy poly(ethylene glycol)-2000 (DMG-PEG) to render the nanoparticle surface hydrophilic and electrostatically neutral. The surface-modified lPEI/DNA nanoparticles showed higher diffusivity and transport in the mucus layer of the GI tract and mediated high levels of transfection efficiency in vitro and in vivo.

Moreover,

these modified nanoparticles demonstrated high levels of GLP-1 expression for more than 24 hours in the liver, lung and intestine in a T2D murine model after a single dose, as well as controlled blood glucose levels within a normal range for at least 18 hours with repeatable therapeutic effects upon multiple dosages. Taken together, this work demonstrates the feasibility of an oral plasmid DNA delivery approach in the treatment of T2D through a facile surface modification to improve the mucus permeability and delivery efficiency of the nanoparticles. KEYWORDS: oral delivery, nucleic acid therapy, GLP-1, type II diabetes, surface coating


Page 3 of 28 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


INTRODUCTION Oral administration of glucagon-like peptide 1 (GLP-1) and its receptor agonists including exendin-4 and liraglutide have become a promising new strategy to treat type II diabetes (T2D) considering its satisfactory patient compliance and low risk of infection.1-3 However, short half-life, immunogenicity, and low oral bioavailability of the protein drugs have compelled researchers to explore new strategies to enhance therapeutic efficiency,4-5 among which gene therapy may offer unique advantages. Compared to peptide or protein therapeutics, nucleic acid has higher stability during the formulation process, lower immunogenicity, and more persistent presence of therapeutic protein as a result of gene expression in local tissue after administration.6-8 However, enzymatic degradation, particularly in the gastric cavity, entrapment in the mucus layer, and low efficiency in crossing the epithelial barrier, all lead to low bioavailability and remain major challenges to overcome for successful oral gene delivery to the target organs.9-12 Penetrating the mucus layer in gastrointestinal (GI) tract presents a common issue for drug and gene delivery. Negatively charged mucin secreted by the epithelium can effectively trap positively charged polycation/DNA complexes via electrostatic interaction, thus hampering the effective transport and delivery to either cellular types in GI tract or other tissues following the crossing of the epithelium, and greatly reducing the transfection efficiency.10 Previous reports have demonstrated that nanoparticles with a hydrophilic and electrostatically neutral surface have higher diffusivity in the mucus layer and higher epithelial cellular uptake and trans-epithelial transport to the blood circulation. For example, hydrophilic grafts including N-(2-hydroxypropyl) methacrylamide (HPMA) and hyaluronic acid (HA) have been coated on cationic nanoparticles to improve oral insulin delivery.13-15 In addition, polyethylene glycol (PEG)-conjugated cationic polymer/DNA nanocomplexes with optimal PEG length and density exhibited high permeability into the mucus layer in mouse lungs and high transgene expression via intratracheal administration. These studies suggest that neutral or negatively charged surfaces on nanoparticles may enhance permeation through various mucosal layers.16, 17 Cationic polymers like linear polyethylenimine (lPEI) have been widely applied in



Page 4 of 28

nucleic acid delivery as a benchmark transfection agent, due to their high transfection efficiency, particularly in vitro.18-20 However, this has not translated into any successful clinical applications due to the cytotoxicity of these polymers and their tendency to aggregate in the physiological environment.21-23 Coating the lPEI/DNA nanocomplexes with lipids or PEGylated lipids has been used to generate lipopolyplexes, and has been shown to reduce the surface charge, improve the colloidal stability, and improve transfection efficiency. Both charged and non-charged lipids have been used to coat onto lPEI/DNA nanocomplexes using non-covalent approaches, typically by mixing and “fusing” blank liposomes with lPEI/DNA nanocomplexes following overnight incubation.24 The liposomes are prepared by hydration/extrusion, reverse-phase evaporation, or the ultrasound dispersion method.24-27 The coated lipopolyplexes are undesirable, as they are polydisperse, with large sizes (>200 nm) and broad size distributions. In addition, these methods are difficult to scale up, resulting in a lower degree of reproducibility. These drawbacks have greatly hampered their widespread application.28-30 Thus, smaller nanoparticle sizes (sub-100 nm) are preferred for higher diffusivity and a more efficient mucus trafficking process.30 However, it is generally difficult to control the size of plasmid DNA nanoparticles to this range in a reproducible manner using conventional preparation methods.31 We have recently developed a technique for continuous production of discrete polyelectrolyte complex nanoparticles, termed flash nanocomplexation (FNC), and demonstrated its ability to prepare nanoparticles with controlled size and high uniformity for the encapsulation of plasmid DNA, protein therapeutics, and small molecular drugs, without the need for complicated formulation processing steps such as sonication, high-pressure homogenization, etc.13, 31-33 This method can also be used for surface coating of nanoparticles.34-35 Moreover, this FNC method for nanoparticle preparation is a continuous process with high production throughput and high reproducibility, portending its high translational potential. In this study, we adopted the FNC method to prepare nanocomplexes with lPEI and plasmid DNA encoding GLP-1, then non-covalently coated them with a mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine

(DPPC,


a

neutral

lipid)

and

Page 5 of 28 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


1,2-dimyristoyl-rac- glycero-3-methoxy poly(ethylene glycol)-2000 (DMG-PEG) to endow the nanoparticles with a highly hydrophilic and electrostatically neutral surface in a continuous and scalable process. We then tested the hypothesis that such a surface coating could yield higher diffusivity and transport across the mucus layer of GI tract and lead to more efficient gene delivery to the liver. We evaluated the DPPC/DMG-PEG-coated lPEI/DNA nanoparticles with the optimized configurations for their efficiency to mediate GLP-1 expression in the liver, lung and small intestine, and tested the therapeutic efficacy in a T2D murine model following oral administration.

MATERIALS AND METHODS Materials. polyetherimide

1,2-Dihexadecanoyl-sn-glycero-3-phosphocholine (lPEI,

average

molecular

weight

(DPPC), of

20

linear kDa),

1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2k), and Tween 20 were purchased from Sigma-Aldrich. 4-(2-Hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES), thiazolyl blue tetrazolium bromide (MTT), glucose, cholesterol (Chol), and DMSO were purchased from Aladdin. Anti-GLP-1 antibody, anti-GADPH antibody, and anti-mouse antibody were purchased from Abcam. GLP-1 and insulin ELISA kit were purchased from Shanghai Renjie Biotechnology Co. Ltd. Alkaline phosphatase (ALP), aspartate transaminase (AST), alanine aminotransferase (ALT), γ-glutamyl transpeptidase (γ-GT), blood urea nitrogen (BUN), and creatinine (CR) assay kits were purchased from Jiancheng Biotech. Co. Ltd. Green fluorescence protein plasmid (pcDNA3.1 + P2A-eGFP, 6.2 kb) vector was obtained from GenScript Biotech Corp. GLP-1 cDNA was synthesized chemically and inserted into the above vector at the HindIII and XbaI sites. The construction of the plasmid DNA was confirmed by direct sequencing. The purified plasmids were stored at -20 °C. Synthesis and characterization of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles. The plasmid DNA was diluted in 25 mM HEPES buffer to a final concentration of 200 µg/mL and the pH was adjusted to 6.5. The lPEI was dissolved in water at a predetermined



Page 6 of 28

concentration to obtain lPEI/DNA complexes with final nitrogen to phosphate (N/P) ratios ranging from 6 to 16. DPPC and Chol were weighed and dissolved in ethanol to concentrations of 3 and 1.3 mg/mL, respectively, to form DPPC/Chol solution. DPPC, Chol and DMG-PEG were weighed and dissolved in ethanol solution at 3, 1.3, and 1 mg/mL, respectively, to form DPPC-DMG-PEG/Chol solution. The preparation processes were performed on a two-step FNC setup (Fig. 1). With a 2-inlet confined impinging jet (CIJ) device, lPEI solution and plasmid DNA solution were impinged at a flow rate of 25 mL/min to form lPEI/DNA nanoparticles. The obtained lPEI/DNA nanoparticles were introduced into a 3-inlet jet FNC device through Inlets 2 and 3; and the DPPC/Chol or DPPC-DMG-PEG/Chol solution was introduced through Inlet 1 at flow rates of 1, 5, 10, 15, 20, 25 or 30 mL/min (same flow rates for all three inlets) to achieve coating of the lPEI/DNA nanocomplex core with the lipids. The products were dialyzed against water overnight to remove the ethanol. To demonstrate the benefits of the FNC method, a traditional fabrication method involving evaporation rotation, hydration and incubation overnight was also used to coat the lPEI/DNA nanocomplex with DPPC and DMG-PEG.29 The

size

distribution

of

uncoated

lPEI/DNA

nanoparticles

(NP-1),

DPPC/(lPEI/DNA)-coated nanoparticles (NP-2) and DPPC-DMG-PEG/(lPEI/DNA)-coated nanoparticles (NP-3) were determined by a Malvern Zetasizer NanoZS90 (Malvern Instruments Ltd., U.K.) 3 times. Nanoparticle samples were stained by phosphotungstic acid; and the morphology was then examined under a transmission electron microscope (TEM; JEOL JEM-1400 Plus, Japan). In order to study the structure of selected nanoparticle samples, cryo-TEM was used. Briefly, carbon-coated copper grids (Electron Microscopy Services, US) were subjected to plasma treatment (N2 glow discharge) for 40 s before sample loading. The imaging grids were prepared using Vitrobot (FEI, US) with a chamber that was maintained at 95% humidity. An aliquot of 6 μL of the concentrated sample solution was dropped to a treated grid and allowed to sit for 1 min. The grid was then blotted and plunged instantly into a liquid ethane reservoir pre-cooled by liquid nitrogen to produce a thin vitreous ice film on the surface of the grid. Afterward, the grid was transferred to a cryo-holder and kept at liquid nitrogen temperature for no more than 24 h before imaging. The cryo-holder temperature was also maintained at liquid nitrogen


Page 7 of 28 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


temperature during imaging to prevent sublimation of vitrified water. Imaging was performed on an FEI Tecnai 12 Twin Transmission Electron Microscope operating at 100 kV. All images were taken by a 16-bit 2K × 2K FEI Eagle bottom mount camera or a Megaview III wide-angle camera.

Figure 1. Schematic illustration of DNA nanoparticle preparation via FNC and the proposed trafficking steps along the gastrointestinal tract.

Stability of nanoparticles in different pH environments. The stability of tested nanoparticles at pH 2.5 (HCl) and pH 7.4 (0.1 M PBS), which simulate the pH environments in the stomach and small intestine, respectively, was measured using the Zetasizer Nano ZS. Briefly, tested samples were incubated with HCl solution or PBS solution (1:4, v/v) in a shaking (100 rpm) water bath at 37 °C. Aliquots of the mixture were collected for DLS



Page 8 of 28

analysis at 5, 15, 30, 60, 120 and 240 min. Cytotoxicity

and

transfection

efficiency

of

DPPC-DMG-PEG/(lPEI/DNA)

nanoparticles in vitro. All cells used in this study, including 293T (human embryonic kidney cell), A549 (adenocarcinomic human alveolar basal epithelial cell), HepG2 (human hepatocellular cancer cell) and HeLa (human cervical cancer cell) cell lines were purchased from American Type Culture Collection (Manassas, VA). All cell lines were incubated in DMEM media supplemented with penicillin (100 units/mL), streptomycin (50 units/mL) and fetal bovine serum (10%) at 37 °C in a humidified incubator with 5% CO2 atmosphere. To assess the toxicity of the nanoparticles, cells were seeded at a density of 2 × 105 cells/mL in a 96-well plate for 24 h and then treated with serial concentrations of nanoparticles for 72 h. After that, MTT dissolved in PBS was added to each well.

After 4 h

incubation, the medium in each well was then removed and DMSO was added to each well instead. The absorbance of each well at 570 nm was then measured by a micro-plate reader (VSERSA Max). To evaluate the transfection efficiency of the nanoparticles, cells were seeded at a density of 1 × 106 cells/mL in a 24-well plate for 24 h. After that, cells were treated with DPPC-DMG-PEG/(lPEI-DNA) nanoparticles with different N/P ratios (equivalent to 1 µg plasmid DNA per well) and incubated for 24 h. Cells were observed under a fluorescence microscope (Leica, DM28, Germany) then harvested and analyzed via flow cytometry (Life Technology, Carlsbad, USA) to determine the transfection efficiency based on the population of eGFP-positive cells. Animal study. All animals used in this study were purchased from the Animal Experimental Center in Guangzhou (Guangzhou, China). All experiments were performed following a protocol approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University. In vivo fluorescence imaging of absorption study. In order to verify the mucus permeability of DPPC-DMG-PEG/(lPEI/DNA) nanoparticles, in vivo absorption studies in the GI tract were performed as previously described.34 Briefly, Balb/c mice were fasted for


Page 9 of 28 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


8 h and then NP-1, NP-2, or NP-3 (plasmid DNA was labeled with SYTOXTM Orange) were administered via oral gavage. After 2 h, all mice were euthanized and their small intestines were collected and frozen in OCT medium. The intestine tissues were sliced, stained and observed under confocal microscopy (Leica SP8). Transfection efficiency of DPPC-DMG-PEG/(lPEI/DNA) nanoparticles in vivo. To access the general gene expression profile in vivo preliminarily, we then used a reporter luciferase plasmid to monitor the transfection efficiency. Balb/c mice were randomly divided into three groups for the three nanoparticle groups (NP-1, NP-2, NP-3). All nanoparticles carried plasmid DNA encoding luciferase. At 8 h before administration, all mice were fed only with water. The nanoparticles were administered via oral gavage to each group at a dose equivalent to 150 µg DNA per mouse. Gene expression in mice was analyzed by whole-body imaging on an IVIS Spectrum Imaging System (Caliper Life Sciences, Hopkinton, MA) at 12, 24 and 36 h post-administration after intraperitoneal (i.p.) injection of luciferin substrate. At 36 h, mice were euthanized to quantify transgene expression levels using tissue homogenates, according to our previous protocol.31 This process was repeated with a separate set of Balb/c mice, randomly divided into three groups and administered with NP-1, NP-2, and NP-3, this time with plasmid DNA encoding GLP-1. Mice were euthanized at 12 h and 24 post-injection. Organs including liver, lung and small intestine were collected for GLP-1 expression analysis. Therapeutic efficacy of DPPC-DMG-PEG/(lPEI/DNA) nanoparticles in vivo. BKS-Leprem2Cd479/Nju mice, also known as db/db mice, were used as a type II diabetes model.36-37 To test the hypoglycemic effect in vivo, db/db mice were randomly divided into a control group (PBS) and three other groups receiving NP-1, NP-2, or NP-3 with GLP-1 plasmid DNA by oral gavage. Before the experiment, the mice were fed only with water for 8 h. Oral gavage of PBS solution for the control group or nanoparticles with a dose equivalent of 150 μg GLP-1 plasmid DNA per mouse for the experimental group was conducted every other day. A total of three doses were given over the course of 6 days. Mice were given free food access for 6 h at 12 h post nanoparticle dosing. The blood glucose level was monitored every 2 h in the daytime and every 6 h at nighttime by a blood



glucose meter (Johnson & Johnson, ONETOUCH○𝑅 Ultra Vue). After 6-day monitoring, all mice were euthanized, with blood samples and organs (including heart, liver, spleen, lungs, kidneys, stomach, small intestines, and pancreas) collected. Western blot analysis of GLP-1 expression. To evaluate the expression of GLP-1 in target organs after the oral dosage, Western Blot assay was applied to analyze the GLP-1 protein level. Briefly, the collected liver, lung and intestine were homogenized in RIPA buffer. The lysis solution was then collected by centrifugation at 12,000 rpm for 15 min. BCA assay was used to determine the total protein concentration. Samples with 40 µg of total proteins were subjected to electrophoresis in 12% tricine gels. The electrophoresed proteins were then transferred to PVDF membrane for 60 min and then immersed in 5% non-fat milk in PBST buffer for 1 h block. The PVDF membranes were then immersed in anti-GLP-1 antibodies at 1:1000 dilution in PBST at 4 °C overnight. The membrane was then incubated with anti-mouse antibodies conjugated with horseradish peroxidase at 1:2000 dilution for 1 h at room temperature and then washed for 3 times with PBST. Protein bands were visualized on a gel-imaging system using ECL Chemiluminescent Substrate Reagent kit, and the semi-quantitative analysis of GLP-1 expression was conducted on Image J based on grayscale, using GADPH as an internal reference. Immunohistochemical analysis of GLP-1 expression. Immunohistochemical analysis was adopted to verify the expression of GLP-1 in target organs. Briefly, the paraffin slides of organs including liver, lung, and intestine were rehydrated and then immersed in PBST. The antigens were retrieved using a microwave oven for 30 s. Then, endogenous peroxidase was inactivated by covering tissue with 3% hydrogen peroxide for 5 min and the slices were washed in PBST 3 times (5 min each on a shaker). After that, the slides were blocked with 10% goat serum for 1 h and then incubated with anti-GLP-1 antibody and incubated overnight in a humidified chamber (4 °C). After washing, a secondary HRP-conjugated anti-mouse antibody was applied to each slide and incubated for 60 min at room temperature. The slides were then washed, stained with DAB substrate and hematoxylin, and mounted on coverslips using a permanent mounting medium.


Page 10 of 28

Page 11 of 28 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


Determination of GLP-1 and insulin level in plasma. The hypoglycemic efficacy of GLP-1 was mainly achieved via promoting insulin secretion in a glucose-dependent manner. GLP-1 and insulin levels in plasma were monitored during the entire treatment process. The blood samples of db/db mice were collected from the orbital venous plexus at pre-determined time points (12, 24, 36, 48 and 84 h) followed by centrifugation at 3,000 rpm for 10 min. The levels of GLP-1 and insulin in the supernatants were then determined by ELISA kits. Evaluation of liver and kidney function. In order to evaluate the pathological and biochemical changes after the treatment with three dosages (150 µg of plasmid DNA per mouse), the serum samples were collected by centrifugation at 3,000 rpm for 10 min. The levels of AST (aspartate transaminase), AKP (alkaline phosphatase), γ-GT (γ-glutamyl transpeptidase), ALT (alanine transaminase), BUN (blood urea nitrogen), and CRE (creatinine) were determined using corresponding kits. The fixed tissues were sectioned, paraffin embedded, dewaxed, and subjected to the standard H&E staining. The stained sections were observed through optical microscopy to detect the pathological changes of the organs. Statistical analysis. All experiments were carried out in triplicate, with data expressed as mean ± S.D. Comparisons between groups were performed using One-way ANOVA or t-test by GraphPad Prism version 5.0 for Windows (GraphPad Software, USA) and p < 0.05 (*); p < 0.01(**) or p < 0.001(***) were considered statistically significant at different levels.

RESULTS AND DISCUSSION Preparation, optimization and characterization of nanoparticles. A plasmid DNA encoding GLP-1 was first constructed and characterized as shown in Fig. S1. A two-step flash nanocomplexation (FNC) platform was then used to prepare core nanoparticles (lPEI-DNA nanocomplex) and the non-covalent modification of neutral lipid and DMG-PEG.



Under the turbulent mixing condition achieved using a two-inlet CIJ mixer, lPEI/DNA nanoparticles (NP-1) were prepared via electrostatic polyelectrolyte complexation. Size distribution and morphology were analyzed. At a flow rate of 25 mL/min and an N/P ratio of 14, the NP-1 produced was spherical in shape with an average diameter of 51 nm and a positive zeta potential of + 40 mV (Fig. S2). Similar results of NP-1 were obtained at N/P ratios ranging from 6 to 16 (Fig. S2). The as-prepared lPEI/DNA nanoparticles were then coated with either DPPC/Chol or DPPC-DMG-PEG/Chol to form NP-2 and NP-3, respectively, using a three-inlet CIJ mixer. The chamber flow rate during the FNC process at which the three streams of materials were impinged was screened. The size of the coated nanoparticles decreased as the flow rate increased. When the flow rate was 20 mL/min or higher, the size of NP-2 and NP-3 were plateaued at the minimum of around 80 nm and 100 nm, respectively (Fig. 2A, D). Similar results of more uniform coating for NP-3 were obtained for lPEI/DNA nanoparticle cores obtained at N/P ratios ranging from 6 to 16 (Fig. S3). In all following experiments, the flow rate for preparing NP-2 and NP-3 were fixed at 20 mL/min. Based on DLS measurements, both sets of nanoparticles were relatively uniform (Fig. 2B, E). TEM results revealed that NP-2 and NP-3 were spherical in shape and confirmed the sizes determined by DLS measurements (Fig. 2C, F). The rapid turbulent mixing condition achieved by the FNC method enables effective mixing and thus more uniform coating and better size control for the assembled nanoparticles. In contrast, nanoparticle coating achieved by manual mixing according to published protocols showed multimodal size distribution with a lower degree of reproducibility (Fig. S4). These results confirm that the FNC method is a superior alternative for more reproducible production of lipid-coated nanoparticles with higher uniformity. The difference in the staining contrast between NP-2 and NP-3 was likely due to the difference in surface charges (NP-2: +5 mV; NP-3: -1.5 mV). Furthermore, a distinctive core-shell structure was captured by cryo-TEM for NP-3 (Fig. S5). Together with the drastic reduction of surface charge from +40 mV to -1.5 mV, these results strongly suggest the presence of DPPC and DMG-PEG coatings on the lPEI/DNA nanoparticles. To investigate the influence of pH environment on the nanoparticles, we monitored the stability of these nanoparticles in the simulated fluid (HCl


Page 12 of 28

Page 13 of 28 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


solution and PBS solution, pH 2.5 and 7.4). From the DLS results (Table S1), nanoparticles coated with DPPC or DPPC-DMG-PEG exhibited high stability in both fluids without obvious size change, suggesting that they would be able to maintain their size when going through different pH environments along the GI tract.

Figure 2. Characterization of DNA nanoparticles. (A) The influence of flow rate on the size and polydispersity index (PDI) of DPPC/(lPEI/DNA) nanoparticles (NP-2). (B) Size distribution of NP-2 measured by dynamic light scattering (DLS). (C) TEM image of DPPC/(lPEI/DNA) nanoparticles. (D) The influence of flow rate on the size and PDI of DPPC-DMG-PEG/(lPEI/DNA) nanoparticles (NP-3). (E) Size distribution of NP-3 measured by DLS. (F) TEM image of NP-3. Data was shown as mean ± SD (n = 3).

Cytotoxicity

and

transfection

efficiency

of

DPPC-DMG-PEG/(PEI-DNA)

nanoparticles in vitro. The cytotoxicity of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles in vitro was evaluated using the MTT assay. The cell viability of 293T, HepG2, A549 and HeLa cell lines were all above 90% compared to the control group upon 72 h incubation with the nanoparticles at different NP-3 concentrations (from 0.05 to 1.6 mg/mL), indicating the DPPC and DMG-PEG coating significantly decreased the cytotoxicity (Fig. 3A). As previously reported, the N/P ratio is critical to the transfection efficiency of



polycation/DNA complexes in vitro and in vivo.38-39 Therefore, the N/P ratio was optimized on the 293T cell line before applying the nanoparticles to mice. The transfection efficiency of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles in the 293T cell line increased as the N/P ratio increased, and the maximum transfection efficiency (~76% eGFP positive cells) was observed with an N/P ratio of 14 or higher (Figs. 3B and S6). The N/P ratio was fixed at 14 for transfection of the A549 and HeLa cell lines, at which high transfection efficiencies (indicated as green fluorescence) were found in both cell lines (Fig. 3C). Therefore, an N/P ratio of 14 was selected for all following in vivo experiments.

Figure 3. Cytotoxicity and transfection efficiency of nanoparticles in vitro. (A) Relative cell metabolic activity of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles on different cells including 293T, A549, HepG2 and Hela (cells without any treatment were used as the control). (B) The influence of N/P ratio on the transfection efficiency of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles on 293T cells (equivalent of 1 µg plasmid DNA per well and incubation for 24 h at 37 °C). (C) Fluorescence microscopy image of different cells after transfection with DPPC-DMG-PEG/(lPEI-DNA) nanoparticles (equivalent of 1 µg plasmid DNA per well and incubation for 24 h at 37 °C). Data are shown as mean ± SD (n = 3), ***p < 0.001.

Transfection efficiency of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles in vivo. To evaluate the gene expression efficiency following oral administration, we used the above DNA delivery system to package the model plasmid encoding luciferase and tested them in


Page 14 of 28

Page 15 of 28 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


Balb/c mice. As shown by the strong bioluminescence signal in Fig. 4A, the NP-3 group maintained high luciferase expression in the liver, lung, and intestine areas 12 to 24 h post-treatment compared with the NP-1 and NP-2 groups. Semi-quantitative analysis of transgene expression intensity showed that NP-3 exhibited 1.5 times higher signal intensity than that of NP-1 or NP-2 group from 12 h to 24 h post-oral administration (Fig. 4B-D). Furthermore, at 36 h post-oral administration, the bioluminescence signal from the NP-3 group in the lung and liver areas remained significantly higher than the other two groups (Fig. 4A). This finding correlated well with the quantitative analysis of organ homogenates (Fig. 4E-G, Table S2). We had also collected the stomach tissue which was homogenized to determine the transgene expression level through bioluminescence assessments. However, there was only background-level signal, indicating very few transfection activities in the stomach. (Data not shown). Based on these results, we concluded that surface coating of the lPEI/DNA nanocomplexes with hydrophilic electrostatically neutral lipids (DPPC and DMG-PEG) significantly improved transfection efficiency of plasmid DNA via oral delivery.



Page 16 of 28

Figure 4. Transgene expression after single dose of plasmid DNA (encoding luciferase) nanoparticles. (A) Whole-body bioluminescence imaging of Balb/c mice at 12, 24 and 36 h post administration

with

single

dose

of

lPEI-DNA

(NP-1),

DPPC/(lPEI-DNA)

(NP-2)

or

DPPC-DMG-PEG/(lPEI-DNA) (NP-3) nanoparticles. Semi-quantification of bioluminescence signal in the (B) liver area; (C) lung area; (D) small intestine. Bioluminescence per mg of (E) liver; (F) lung; (G) small intestine normalized by organ weight at 36 h; Data is shown as mean ± SD (n = 4), *p < 0.05, **p < 0.01 vs. NP-1 group. # p < 0.05, ##p < 0.01 vs. NP-2 group.

The in vivo target gene expression of GLP-1 in normal Balb/c mice was also assessed after oral delivery of DNA-loaded nanoparticles by Western Blot analysis. NP-2 and NP-3 were found to facilitate a fast onset of transgene activities within 12 h and the expression maintained till at least 24 h (Fig. 5). In contrast, the NP-1 group showed delayed and low levels of GLP-1 expression in the liver, lungs, and intestine. Both sets of transgene


Page 17 of 28 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


expression data on luciferase and GLP-1 indicate that high surface charge of NP-1 may lead to high retention in the mucus layer and higher degrees of enzymatic degradation and clearance through GI tract, whereas NP-2 and NP-3 showed faster transport kinetics across the mucus layer, likely due to the hydrophilic and electrostatically neutral surface of DPPC and DMG-PEG-coated nanoparticles (Fig. S7). The NP-3 group resulted in higher GLP-1 expression in the liver than that of the NP-1 or NP-2 groups 24 h after oral dosing. Additionally, NP-1 had almost no transfection in the lungs, while the transfection efficiency for NP-2 and NP-3 are comparable. However, NP-1, NP-2 and NP-3 resulted in similar levels of transgene activity in the intestines. These results demonstrate that lPEI-DNA nanoparticles coated with DPPC and DPPC-DMG-PEG ensured fast and sustained expression of GLP-1 for at least 24 h, thus serving as the basis for fast-acting and persistent blood glucose control. Based on the results of in vivo imaging and western blot analysis, we speculate that the successful transport of DPPC-DMG-PEG/lPEI-DNA NPs in the mucus layer of gastric and intestinal tissue is mainly attributed to the surface coating of DMG-PEG and DPPC on the nanoparticles. The hydrophilic and neutral surface effectively minimized the hydrophobic interactions and polyvalent adhesive interaction between lPEI-DNA (highly positively charged) and mucus layer (highly negatively charged), thereby promoting the diffusion and penetration of the nanoparticles in mucus layer as they moved through openings between mucin mesh fibers.40 After that, the nanoparticles were transported by intestinal epitheliums into the capillaries. Owing to the decoration of DMG-PEG, the nanoparticles could circulate in vessel stably with minimized clearance of reticuloendothelial system and finally accumulated in the major organs like liver and lungs. Nanoparticles then bound to and entered cells through fusion of the DPPC lipid bilayer with cell membrane. Finally, lPEI-DNA NPs escaped from endosome due to the proton sponge effect of lPEI following by DNA release, nuclear transport, insertion and subsequent transcription and translation.41



Figure 5. GLP-1 expression after oral administration of DNA (encoding GLP-1) nanoparticles in Balb/c mice. This was quantified in the (A) liver, (B) lungs, and (C) intestine. The semi-quantitative analysis of GLP-1 expression level was measured in the (D) liver, (E) lungs, and (F) intestine. Data is shown as mean ± SD (n = 3).

Therapeutic efficacy of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles in vivo. To evaluate therapeutic efficacy of the DPPC-DMG-PEG/(lPEI-DNA) nanoparticles in a T2D murine model, the blood glucose level (BGL) was monitored throughout the experiment after oral gavage of the nanoparticles. The BGL of mice treated by NP-2 and NP-3 dropped into the normal BGL range (5~10 mmol/L) at 6 h after dosing (Fig. 6A). With a glucose surge due to food intake during the feeding period, the BGL of the NP-3 group increased to only 16 mmol/L (~60% of the original level), then quickly lowered to the normal range within 4 h and remained at about 8 mmol/L during the fasting period. The higher efficiency of the NP-3 group in maintaining BGL in comparison with other groups suggests that GLP-1 is a safe hypoglycemic peptide for BGL control in a glucose-dependent manner. The therapeutic effect of NP-3 was consistent following the second dose and the feeding/fasting cycle. After the third dose, the BGL of mice treated with NP-3 was still 50% lower than that of mice in the control group, even after 36 h of free feeding. The NP-2 group had a similar BGL control efficacy as the NP-3 group in reducing the BGL to the normal range before the first feeding, but, was not as effective as NP-3 in maintaining the normal BGL during the feeding and fasting periods. In contrast, the BGL of mice dosed by NP-1 could not reach the normal blood glucose concentration range during the entire experimental period, presenting a similar


Page 18 of 28

Page 19 of 28 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


uncontrolled BGL profile as the control group. The semi-quantitative analysis of the BGL represented as the area under the curve (AUC) of Fig. 6B reinforces the efficacy ranking of the nanoparticles as NP-3 > NP-2 > NP-1, in terms of the effectiveness in reducing and maintaining BGL. Interestingly, we noticed that the BGL from the NP-2 and NP-3 groups decreased to about 13 mmol/L just after 6 h post administration. Therefore, a drop of the blood glucose, a result of gene expression starting as early as 6 h post oral gavage, was confirmed by dosing nanoparticles with luciferase reporter plasmid construct and IVIS live imaging. At 6 h, transgene activities were detectable (Fig. S8). In addition, the reduction of the body weight from all treatment groups further verified the expression profile of GLP-1 considering its effect on body weight control (Fig. 6C). The local GLP-1 levels in the major organs and blood GLP-1 concentrations were determined by western blot and ELISA. The results showed significantly higher local GLP-1 expression in the liver and blood in the NP-3 group during the entire treatment than that of the NP-1 or NP-2 groups (Fig. 6D-F). Correspondingly, the secretion of insulin from the NP-3 group increased to the highest level (~32 mIU/L) at 24 h and maintained this level until 48 h post-administration, which matched the time vs. concentration profile of GLP-1 (Fig. 6F-G) and demonstrated the most effective BGL control in NP-3 group. Immunohistochemical analysis of GLP-1 expression was also performed and the results agreed with the high transfection efficiency of NP-3 in the liver (Fig. 7). In the NP-2 and NP-3 groups, appreciable GLP-1 expression was also detected in the lungs and intestines. Liver and kidney dysfunction usually accompanies the progression of diabetes,42-43 leading to abnormalities in several blood biochemical indicators. After treatment by NP-3, the elevated levels of AST, ALT, CRE and BUN were all reduced significantly (Fig. 6H-K). Our results clearly demonstrate that the benchmark lPEI/DNA nanoparticles decorated by DPPC and DMG-PEG have a higher oral gene delivery and transfection efficiency while improving liver and kidney functions. These results collectively suggest the potential of this system in treating T2D through plasmid DNA encoding GLP-1.



Figure 6. Therapeutic efficiency of DNA nanoparticles after oral administration in a T2D mouse (db/db) model. (A) Blood glucose level vs. time profiles of type II diabetes mice, following multiple oral dosages of different formulations (150 µg DNA per time). p.o.: per os, oral feeding. (B) The area under curve (AUC) of the blood glucose level vs. time profiles. (C) The body weight change from different groups during the treatment (1~6 days). (D) The western blotting analysis of GLP-1 expression in db/db mice organs including liver, lung and intestine. (E) The semi-quantitative analysis of western blotting results. (F) The level of GLP-1 in plasma from different treatment group during the treatment (12~84 h). (G) The level of insulin in plasma from different treatment group during the treatment (12~84 h). The enzyme activities of (H) AST (I) ALT in liver after treatment and (J) BUN (K) Cr in kidney after the treatment. Control group: db/db model mice without treatment. *p < 0.05, **p < 0.01 and ***p < 0.001 vs. control group. ###p < 0.001 vs.NP-2 group. Data is shown as mean ± SD (n = 5).


Page 20 of 28

Page 21 of 28 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


Figure 7. The immunohistochemical analysis of GLP-1 expression in db/db mouse liver, lung, and intestine. GLP-1 peptide-positive areas are indicated in brown. (Scale bar = 50 µm).

In vivo toxicity study of DPPC-DMG-PEG/(lPEI-DNA) nanoparticles. The toxicity was evaluated by monitoring the blood biochemical indicators and histological assessments through H&E staining to organ sections. Among all indicators tested, there were no findings on tissue toxicity, suggesting that DPPC-DMG-PEG/(lPEI-DNA) nanoparticles did not induce acute toxicity to the liver and kidney (Fig. S9). Furthermore, as seen in the liver, the hepatocytes were significantly swollen with regions of lytic necrosis found in the liver in the model control group. In contrast, the pathological damages in liver were alleviated from all treatment groups especially NP-3 group after multiple dosages, indicating the sustained expression of GLP-1 could relieve the impairment of liver function while controlling the BGL effectively. Aside from that, there was no significant pathological change observed in the heart, spleen, lungs, kidneys, stomach, intestines or pancreas (Fig. 8). Therefore, the DPPC-DMG-PEG/(lPEI-DNA) nanoparticles are considered biocompatible after multiple oral dosages.



Figure 8. The pathological study after treatment of DNA nanoparticles. (Scale bar = 100 µm)

CONCLUSIONS We have successfully constructed an oral gene delivery system with a hydrophilic and electrostatically neutral surface using the two-step FNC process. The lipid-decorated DNA nanoparticles exhibited faster mucus permeability and trans-cellular transport efficiency in the small intestine. After a single oral dose, sustained expression of GLP-1 was detected in the liver, lung, and small intestine 12 to 36 h post administration. BGL was promptly reduced to the normal range in 6 h following oral dose and maintained within the normal range (5–10 mmol/L) for at least 18 h in a T2D murine model. In addition, the BGL only increased to 60% of the original level (16 mmol/L) with glucose intake and recovered to the normal range in a few hours. Similar glucose control effect was observed upon repeated dosing without negatively affecting liver and kidney functions. By benefitting from the scalability and reproducibility of FNC, we anticipate that this oral gene delivery system holds great translational potential in the treatment of T2D. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: ############.


Page 22 of 28

Page 23 of 28 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


AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] (Y. Chen) * E-mail: [email protected] (H. Mao) * E-mail: [email protected]. (Z. He) Author Contributions §These

authors contributed equally to this work.

Author Contributions Y.C., T.N., Z.H., L.L., K.W.L. and H.-Q.M. conceived and planned the study. T.N., J.Z., K.C., Y.Z. and Z.H. conducted all experiments and performed data analysis and interpretation. Y.C., L.L., K.W.L. and H.-Q.M. also participated in data analysis and result interpretation. T.N., Y.H., Z.H., K.C., Y.C., K.W.L. and H.-Q.M. wrote the manuscript, which was reviewed and edited by all co-authors. ACKNOWLEDGEMENTS This work was supported by the Guangdong Innovative and Entrepreneurial Research Team Program (No. 2013S086), Natural Science Foundation of Guangdong Province (No. 2014A030312018), and Natural Science Foundation of China (No. 51820105004). Notes The authors declare no competing financial interests.



REFERENCES (1) Lovshin, J. A.; Drucker, D. J. Incretin-based Therapies for Type 2 Diabetes Mellitus. Nature Reviews Endocrinology 2009, 5 (5), 262. (2) Zinman, B.; Gerich, J.; Buse, J. B.; Lewin, A.; Schwartz, S.; Raskin, P.; Hale, P. M.; Zdravkovic, M.; Blonde, L.; Investigators, L.-S. Efficacy and Safety of the Human Glucagon-like Peptide-1 Analog Liraglutide in Combination with Metformin and Thiazolidinedione in Patients with Type 2 Diabetes (LEAD-4 Met+ TZD). Diabetes Care 2009, 32 (7), 1224-1230. (3) Meier, J. J. GLP-1 Receptor Agonists for Individualized Treatment of Type 2 Diabetes Mellitus. Nature Reviews Endocrinology 2012, 8 (12), 728. (4) Hui, H.; Farilla, L.; Merkel, P.; Perfetti, R. The Short Half-life of Glucagon-like Peptide-1 in Plasma does not Reflect its Long-lasting Beneficial Effects. European Journal of Endocrinology 2002, 146 (6), 863-869. (5) Beglinger, C.; Poller, B.; Arbit, E.; Ganzoni, C.; Gass, S.; Gomez-Orellana, I.; Drewe, J. Pharmacokinetics and Pharmacodynamic Effects of Oral GLP-1 and PYY3-36: a Proof-of-concept Study in Healthy Subjects. Clinical Pharmacology & Therapeutics 2008, 84 (4), 468-474. (6) Hatakeyama, H.; Akita, H.; Harashima, H. The Polyethyleneglycol Dilemma: Advantage and Disadvantage of PEGylation of Liposomes for Systemic Genes and Nucleic Acids Delivery to Tumors. Biological and Pharmaceutical Bulletin 2013, 36 (6), 892-899. (7) Zheng, D.; Giljohann, D. A.; Chen, D. L.; Massich, M. D.; Wang, X.-Q.; Iordanov, H.; Mirkin, C. A.; Paller, A. S. Topical Delivery of SiRNA-based Spherical Nucleic Acid Nanoparticle Conjugates for Gene Regulation. Proceedings of the National Academy of Sciences 2012, 109 (30), 11975-11980. (8) Zhu, G.; Ye, M.; Donovan, M. J.; Song, E.; Zhao, Z.; Tan, W. Nucleic Acid Aptamers: an Emerging Frontier in Cancer Therapy. Chemical communications 2012, 48 (85), 10472-10480. (9) Singh, R.; Singh, S.; Lillard, J. W. Past, Present, and Future Technologies for Oral Delivery of Therapeutic Proteins. Journal of Pharmaceutical Sciences 2008, 97 (7), 2497-2523. (10) Blanchette, J.; Kavimandan, N.; Peppas, N. A. Principles of Transmucosal Delivery of Therapeutic Agents. Biomedicine & Pharmacotherapy 2004, 58 (3), 142-151. (11) Le, Z.; Chen, Y.; Han, H.; Tian, H.; Zhao, P.; Yang, C.; He, Z.; Liu, L.; Leong, K. W.; Mao, H.-Q. Hydrogen-Bonded Tannic Acid-Based Anticancer Nanoparticle for Enhancement of Oral Chemotherapy. ACS Applied Materials & Interfaces 2018, 10 (49), 42186-42197. (12) Ensign, L. M.; Cone, R.; Hanes, J. Oral Drug Delivery with Polymeric Nanoparticles: the Gastrointestinal Mucus Barriers. Adv Drug Deliv Rev 2012, 64 (6), 557-70. (13) He, Z.; Santos, J. L.; Tian, H.; Huang, H.; Hu, Y.; Liu, L.; Leong, K. W.; Chen, Y.; Mao, H.-Q. Scalable Fabrication of Size-controlled Chitosan Nanoparticles for Oral Delivery of Insulin. Biomaterials 2017, 130, 28-41. (14) Liu, M.; Zhang, J.; Zhu, X.; Shan, W.; Li, L.; Zhong, J.; Zhang, Z.; Huang, Y. Efficient Mucus Permeation and Tight Junction Opening by Dissociable "Mucus-inert" Agent Coated Trimethyl Chitosan Nanoparticles for Oral Insulin Delivery. Journal of Controlled Release : official journal of the Controlled Release Society 2016, 222, 67-77.


Page 24 of 28

Page 25 of 28 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


(15) Shan, W.; Zhu, X.; Liu, M.; Li, L.; Zhong, J.; Sun, W.; Zhang, Z.; Huang, Y. Overcoming the Diffusion Barrier of Mucus and Absorption Barrier of Epithelium by Self-assembled Nanoparticles for Oral Delivery of Insulin. ACS Nano 2015, 9 (3), 2345-2356. (16) Tang, B. C.; Dawson, M.; Lai, S. K.; Wang, Y.-Y.; Suk, J. S.; Yang, M.; Zeitlin, P.; Boyle, M. P.; Fu, J.; Hanes, J. Biodegradable Polymer Nanoparticles that Rapidly Penetrate the Human Mucus Barrier. Proceedings of the National Academy of Sciences 2009, 106 (46), 19268-19273. (17) Suk, J. S.; Kim, A. J.; Trehan, K.; Schneider, C. S.; Cebotaru, L.; Woodward, O. M.; Boylan, N. J.; Boyle, M. P.; Lai, S. K.; Guggino, W. B. Lung Gene Therapy with Highly Compacted DNA Nanoparticles that Overcome the Mucus Barrier. Journal of Controlled Release 2014, 178, 8-17. (18) Akinc, A.; Thomas, M.; Klibanov, A. M.; Langer, R. Exploring Polyethylenimine-mediated DNA Transfection and the Proton Sponge Hypothesis. The Journal of Gene Medicine: A Cross-disciplinary Journal for Research on the Science of Gene Transfer and its Clinical Applications 2005, 7 (5), 657-663. (19) Yue, Y.; Jin, F.; Deng, R.; Cai, J.; Dai, Z.; Lin, M. C.; Kung, H.-F.; Mattebjerg, M. A.; Andresen, T. L.; Wu, C. Revisit Complexation Between DNA and Polyethylenimine—Effect of Length of Free Polycationic Chains on Gene Transfection. Journal of Controlled Release 2011, 152 (1), 143-151. (20) Roy, K.; Mao, H.-Q.; Huang, S.-K.; Leong, K. W. Oral Gene Delivery with Chitosan–DNA Nanoparticles Generates Immunologic Protection in a Murine Model of Peanut Allergy. Nature Medicine 1999, 5 (4), 387. (21) Parhamifar, L.; Larsen, A. K.; Hunter, A. C.; Andresen, T. L.; Moghimi, S. M. Polycation Cytotoxicity: a Delicate Matter for Nucleic Acid Therapy—Focus on Polyethylenimine. Soft Matter 2010, 6 (17), 4001-4009. (22) Lv, H.; Zhang, S.; Wang, B.; Cui, S.; Yan, J. Toxicity of Cationic Lipids and Cationic Polymers in Gene Delivery. Journal of Controlled Release 2006, 114 (1), 100-109. (23) Lin, P. Y.; Chiu, Y. L.; Huang, J. H.; Chuang, E. Y.; Mi, F. L.; Lin, K. J.; Juang, J. H.; Sung, H. W.; Leong, K. W. Oral Nonviral Gene Delivery for Chronic Protein Replacement Therapy. Advanced Science 2018, 5 (8), 1701079. (24) Lehtinen, J.; Hyvonen, Z.; Subrizi, A.; Bunjes, H.; Urtti, A. Glycosaminoglycan-resistant and pH-sensitive Lipid-coated DNA Complexes Produced by Detergent Removal Method. Journal of Controlled Release : official journal of the Controlled Release Society 2008, 131 (2), 145-149. (25) Mukherjee, A.; Waters, A. K.; Kalyan, P.; Achrol, A. S.; Kesari, S.; Yenugonda, V. M. Lipid-polymer Hybrid Nanoparticles as a Next-generation Drug Delivery Platform: State of the Art, Emerging Technologies, and Perspectives. International Journal of Nanomedicine 2019, 14, 1937-1952. (26) Song, H.; Wang, G.; He, B.; Li, L.; Li, C.; Lai, Y.; Xu, X.; Gu, Z. Cationic Lipid-coated PEI/DNA Polyplexes with Improved Efficiency and Reduced Cytotoxicity for Gene Delivery into Mesenchymal Stem Cells. International Journal of Nanomedicine 2012, 7, 4637-4648. (27) Luo, X.; Feng, M.; Pan, S.; Wen, Y.; Zhang, W.; Wu, C. Charge Shielding Effects on Gene Delivery of Polyethylenimine/DNA Complexes: PEGylation and Phospholipid coating. Journal of Materials Science. Materials in Medicine 2012, 23 (7), 1685-1695. (28) Schafer, J.; Hobel, S.; Bakowsky, U.; Aigner, A. Liposome-polyethylenimine Complexes for Enhanced DNA and SiRNA Delivery. Biomaterials 2010, 31 (26), 6892-6900. (29) Ewe, A.; Panchal, O.; Pinnapireddy, S. R.; Bakowsky, U.; Przybylski, S.; Temme, A.; Aigner, A. Liposome-polyethylenimine Complexes (DPPC-PEI Lipopolyplexes) for Therapeutic SiRNA Delivery in vivo.



Nanomedicine : Nanotechnology, Biology, and Medicine 2017, 13 (1), 209-218. (30) Xuan, M.; Shao, J.; Lin, X.; Dai, L.; He, Q. Self-propelled Janus Mesoporous Silica Nanomotors with Sub-100 nm Diameters for Drug Encapsulation and Delivery. Chemphyschem : a European Journal of Chemical Physics and Physical Chemistry 2014, 15 (11), 2255-2260. (31) He, Z.; Hu, Y.; Nie, T.; Tang, H.; Zhu, J.; Chen, K.; Liu, L.; Leong, K. W.; Chen, Y.; Mao, H. Q. Size-controlled Lipid Nanoparticle Production Using Turbulent Mixing to Enhance Oral DNA Delivery. Acta Biomaterialia 2018, 81, 195-207. (32) He, Z.; Hu, Y.; Gui, Z.; Zhou, Y.; Nie, T.; Zhu, J.; Liu, Z.; Chen, K.; Liu, L.; Leong, K. W.; Cao, P.; Chen, Y.; Mao, H. Q. Sustained Release of Exendin-4 from Tannic Acid/Fe (III) Nanoparticles Prolongs Blood Glycemic Control in a Mouse Model of Type II Diabetes. Journal of Controlled Release : official journal of the Controlled Release Society 2019, 301, 119-128. (33) Qiao, D.; Liu, L.; Chen, Y.; Xue, C.; Gao, Q.; Mao, H.-Q.; Leong, K. W.; Chen, Y. Potency of a Scalable Nanoparticulate Subunit Vaccine. Nano Letters 2018, 18 (5), 3007-3016. (34) He, Z.; Liu, Z.; Tian, H.; Hu, Y.; Liu, L.; Leong, K. W.; Mao, H.-Q.; Chen, Y. Scalable Production of Core– shell Nanoparticles by Flash Nanocomplexation to Enhance Mucosal Transport for Oral Delivery of Insulin. Nanoscale 2018, 10 (7), 3307-3319. (35) Tian, H.; He, Z.; Sun, C.; Yang, C.; Zhao, P.; Liu, L.; Leong, K. W.; Mao, H. Q.; Liu, Z.; Chen, Y. Uniform Core-Shell Nanoparticles with Thiolated Hyaluronic Acid Coating to Enhance Oral Delivery of Insulin. Advanced Healthcare Materials 2018, 7 (17), e1800285. (36) Srinivasan, K.; Ramarao, P. Animal Model in Type 2 Diabetes Research: An Overview. Indian Journal of Medical Research 2007, 125 (3), 451. (37) Chen, D.; Wang, M. W. Development and Application of Rodent Models for Type 2 Diabetes. Diabetes, Obesity and Metabolism 2005, 7 (4), 307-317. (38) Mansouri, S.; Lavigne, P.; Corsi, K.; Benderdour, M.; Beaumont, E.; Fernandes, J. C. Chitosan-DNA Nanoparticles as Non-viral Vectors in Gene Therapy: Strategies to Improve Transfection Efficacy. European Journal of Pharmaceutics and Biopharmaceutics 2004, 57 (1), 1-8. (39) Intra, J.; Salem, A. K. Characterization of the Transgene Expression Generated by Branched and Linear Polyethylenimine-plasmid DNA Nanoparticles in vitro and After Intraperitoneal Injection in vivo. Journal of Controlled Release 2008, 130 (2), 129-138. (40) Lai, S. K.; Wang, Y. Y.; Hanes, J. Mucus-penetrating Nanoparticles for Drug and Gene Delivery to Mucosal Tissues. Adv Drug Deliv Rev 2009, 61 (2), 158-171. (41) Buck, J.; Grossen, P.; Cullis, P. R.; Huwyler, J. r.; Witzigmann, D. Lipid-based DNA Therapeutics: Hallmarks of Non-viral Gene Delivery. ACS Nano 2019, 13 (4), 3754-3782. (42) Yamabe, N.; Noh, J. S.; Park, C. H.; Kang, K. S.; Shibahara, N.; Tanaka, T.; Yokozawa, T. Evaluation of Loganin, Iridoid Glycoside from Corni Fructus, on Hepatic and Renal Glucolipotoxicity and Inflammation in Type 2 Diabetic db/db Mice. European Journal of Pharmacology 2010, 648 (1-3), 179-187. (43) Soto-Urquieta, M. G.; López-Briones, S.; Pérez-Vázquez, V.; Saavedra-Molina, A.; González-Hernández, G. A.; Ramírez-Emiliano, J. Curcumin Restores Mitochondrial Functions and Decreases Lipid Peroxidation in Liver and Kidneys of Diabetic db/db Mice. Biological Research 2014, 47 (1), 74.


Page 26 of 28

Page 27 of 28 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




208x174mm (300 x 300 DPI)


Page 28 of 28

Surface Coating Approach to Overcome Mucosal Entrapment of DNA

Recommend Documents