Preparation and Characterization of a Liver Targeted, Poly(amidoamine) Based, Gene Delivery System

Abstract

Nonalcoholic steatohepatitis (NASH) is an aggressive liver disease that is considered a major cause of liver cirrhosis and hepatocellular carcinoma. NASH is characterized by multiple underlying genetic mutations, with no approved cure to date. Gene therapies that target those genetic mutations may play a major role in treating this disease, once delivered specifically to the hepatocytes. In this chapter we present, in detail, the synthesis and the characterization of an efficient gene delivery system capable of targeting hepatocytes by exploiting the overexpression of asialoglycoprotein receptors on their cell surface. The targeting ligand, galactose derivative, lactobionic acid (Gal), is first conjugated to bifunctional poly(ethylene glycol) (PEG), and then the formed PEG-Gal is further conjugated to the positively charged polymer, Poly(amidoamine) (PAMAM) to form a PAMAM-PEG-Gal construct that can complex and deliver genetic material (e.g. pDNA, siRNA, mRNA) specifically to hepatocytes. We first synthesize PAMAM-PEG-Gal using carbodiimide click chemistry. The synthesized conjugate is characterized using ¹H NMR spectroscopy and mass spectrometry. Next, nanoplexes are prepared by combining the positively charged conjugate and the negatively charged genetic material at different nitrogen to phosphate (N/P) ratios; then the size, charge, electrophoretic mobility and surface morphology of those nanoplexes are estimated. The simplicity of complexing our conjugate with any type of genetic material, and the ability of our delivery system to overcome the current limitations of delivering naked genetic material, and the efficiency of delivering its payload specifically to hepatocytes, makes our formulation a promising tool to treat any type of genetic abnormality that arises in hepatocytes, and specifically NASH.

1. Introduction

A large proportion of liver diseases arise from genetic abnormalities in hepatocytes [1]. Nonalcoholic steatohepatitis (NASH) is an example of those genetic disorders [2], that is characterized by excessive accumulation of fats in the hepatocytes, hepatocyte injury, inflammation and fibrosis [3]. NASH has been listed as a major cause of liver cirrhosis and hepatocellular carcinoma [4]. Unfortunately, there are no permanent cures for such genetic disorder, and the current therapeutic interventions tend to only alleviate the symptoms, leaving liver transplantation as the only available cure [5,6]. Gene therapies play a major role in overcoming such therapeutic limitations [7], where delivering appropriate genetic material to the mutated hepatocytes can result in remedying this genetic disorder [8]. However, utilizing naked genetic material as a therapy is limited, because once administered intravenously, they are prone to accelerated clearance by the kidney [9], rapid degradation by nucleases in the circulation [10], and reduced cellular uptake by hepatocytes [11]. In this chapter, we describe the synthesis and characterization of a well-designed gene delivery system capable of delivering any type of genetic material with great specificity to hepatocytes.

Poly(amidoamine) (PAMAM) is a positively charged dendrimer made of repetitive branched amine and amide containing groups, that has the ability to complex with, and condense negatively charged genetic material into small nanometer sized nanoplexes [12]. The formed nanoplexes are capable of protecting the loaded genetic material subsequent to IV administration until they reach their target site. They mainly protect their loaded cargo from any possible degradation by nucleases present throughout the whole body, in addition to protecting them from harsh endosomal conditions, once they reach their target site, through their ability to induce endosomal escape [13]. Unfortunately, the presence of large numbers of surface amine groups on PAMAM molecules results in a toxic interaction with RBCs membranes, causing their hemolysis [14]. Polyethylene glycol (PEG) is a widely used and safe hydrophilic polymer that, once conjugated to some of the surface amine groups of PAMAM, can decrease the charge density and reduce direct interactions with RBCs through steric hindrance [15,16]. The advantages gained from the addition of PEG to PAMAM surface, come with the disadvantage of a significant reduction in hepatocyte uptake, and thus reduced therapeutic activity [17,18]. Addition of a targeting moiety that can guide the PEGylated nanoplex to specifically interact with hepatocytes could counter this drawback. Galactose is chosen as our targeting moiety because it can bind specifically to asialoglycoprotein receptors [19], initiating receptor-mediated endocytosis. Asialoglycoprotein receptors are expressed selectively on hepatocytes, both normal and cancerous [20], rendering them a potential target for liver disease therapeutics.

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC)/N-hydroxy sulfosuccinimide (sulfo-NHS) click chemistry is one of the most common and simple chemistries used to form covalent amide bonds between carboxy and amine terminal groups [21]. We utilize this EDC/sulfo-NHS chemistry to carry out our conjugation. We use bifunctional PEG molecules with both amine and carboxyl end groups. The amine group of PEG is first conjugated to the activated carboxylic group of a disaccharide derivative of galactose named lactobionic acid (Gal). Next, the free carboxylic group of the formed PEG-Gal is further conjugated to PAMAM amine groups to form our final conjugate, PAMAM-PEG-Gal. The synthesized conjugate is evaluated using ¹H NMR spectroscopy to confirm the conjugation process and to calculate the exact molar ratios of the conjugate components and eventually the molecular weight (Mw) of our final conjugate. Moreover, mass spectrometry is also carried out to confirm the presence of Gal in our final conjugate. Next, we prepare nanoplexes by combining PAMAM-PEG-Gal and plasmid DNA (pDNA) at different nitrogen to phosphate ratios (N/P). Finally, we characterize the prepared nanoplexes by measuring their size and charge, where positively charged, nanometer-sized nanoplexes indicate successful fabrication. The prepared nanoplexes should be able to retain their cargo while circulating throughout the body, and also release their cargo once taken up by the target cells. Thus, gel electrophoresis is performed on the nanoplexes in order to estimate the ability of the nanoplexes to complex pDNA and avoid the plasmid being released and migrating into the gel. We also assess the ability of the nanoplexes to release their cargo once pretreated with heparin salt, which imitates the conditions following cellular uptake. Surface morphology is evaluated using transmission electron microscopy, where spherical to irregular shaped nanometer structures indicate successful fabrication of the nanoplexes. In conclusion, we propose that our synthesized conjugate has the potential to serve as a universal gene delivery system to treat NASH, as well as any other liver disease with underlying genetic abnormality.

2. Materials

2.1. Synthesis of PAMAM-PEG-Gal:

1. Nanopure water (NANOpure Diamond™, Barnstead International) or any other source of sterile water.

2. Sodium hydroxide (NaOH).

3. 4-Morpholineethanesulfonic acid (MES) buffer.

4. Lactobionic acid (Gal) (Sigma-Aldrich).

5. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (ThermoFisher).

6. N-Hydroxy sulfosuccinimide (Sulfo-NHS) (ThermoFisher).

7. 2-Mercaptoethanol.

8. Poly(ethylene glycol) 2-aminoethyl ether acetic acid (PEG) (Sigma-Aldrich).

9. Poly(amidoamine) dendrimer (PAMAM), ethylenediamine core, generation 5.0 (G5) solution, 5 wt % in methanol, density of 0.797 g/mL (Sigma-Aldrich).

10. Thirty mL and 2 L Pyrex® glass beakers.

11. Millex-GV syringe filter unit, 0.22 μm, PVDF, 33 mm, gamma sterilized.

12. Spectra/Por® 7 dialysis membrane, 1K MWCO, 45 mm.

13. SnakeSkin™ dialysis tubing, 10K MWCO, 22 mm.

14. FreeZone 4.5 L Benchtop Freeze Dry System (Labconco Corporation).

2.2. Characterization of PAMAM-PEG-Gal:

1. ¹H NMR spectroscopy.

2. Mass spectrometry.

2.3. Preparation of Nanoplexes of PAMAM-PEG-Gal and pDNA:

1. DNAse and RNAse Free, Ultrapure Distilled Water.

2. Enhanced Green Fluorescent Protein plasmid DNA (pDNA) provided as bacteria in an agar stab (Addgene), or any other available pDNA.

3. Two-mL microcentrifuge tubes.

4. Nanodrop 200 spectrophotometer (ThermoFisher).

2.4. Characterization of Nanoplexes of PAMAM-PEG-Gal and pDNA:

2.4.1. Size and Surface Charge Measurements:

1. Freshly prepared nanoplexes.

2. Zetasizer Nano ZS (Malvern Instrument Ltd).

2.4.2. Gel Electrophoresis:

1. FisherBiotech™ Horizontal Electrophoresis Systems (Fisher Scientific).

2. Agarose.

3. Ethidium bromide solution, 10 mg/mL.

4. Tris-Borate-EDTA buffer 10X solution: 0.89 M tris, 0.89 M borate, and 0.02 M EDTA.

5. 10x Blue Juice.

6. TrackIt™ 1 kb Plus DNA ladder.

7. Heparin sodium salt (Sigma-Aldrich).

8. iBright FL1000 Imaging System (Invitrogen).

2.4.3. Transmission Electron Microscope (TEM) Analysis:

1. TEM copper grids 200 mesh covered with Formvar and coated with carbon (Ted Pella).

2. Phosphotungstic acid hydrate.

3. JEOL JEM-1230 TEM equipped with a Gatan UltraScan 1000 2k × 2k CCD acquisition system.

3. Methods

3.1. Synthesis of PAMAM-PEG-Gal:

3.1.1. Buffers and stock solutions.

1. Prepare 1 N NaOH solution by dissolving 4 g of NaOH in 100 mL of nanopure water (see Note 1).

2. Prepare 0.1 M MES buffer solution by dissolving 4.67 g of MES in 239 mL of nanopure water; raise the final pH to 6 by adding 10.5 mL of 1 N NaOH, and then store at 4°C.

3.1.2. Conjugation of the Gal carboxylic group to the PEG amine group to form PEG-Gal.

In the first step, the free carboxylic group of Gal is activated through EDC/sulfo-NHS, and then the activated Gal-NHS forms an amide bond with the free PEG amine group.

1. Remove PEG, EDC, sulfo-NHS, and MES buffer from fridge/freezer and let them reach room temperature before use (see Note 2).

2. Add 20 mL of MES buffer to a 30-mL glass beaker and transfer the beaker to a magnetic stirrer and start stirring the solution.

3. Weigh out 85.325 mg (238 μmoles) Gal, transfer to the beaker and stir for 5 min until the solution becomes clear (see Note 3).

4. Weigh out 45.65 mg (238 μmoles) EDC and 129.25 mg Sulfo-NHS (595 μmoles), transfer both to the same 2 mL micro-centrifuge tube, add 1 mL of MES buffer and then vortex for 2 min until the solution becomes clear (see Note 3).

5. Add the EDC and sulfo-NHS solution dropwise to the beaker containing the Gal solution while stirring, in order to form NHS ester of Gal (Gal-NHS).

6. Stir the solution for 15 min, and then add 168 μL (2380 μmoles) of 2-mercaptoethanol under a fume hood (see Note 4).

7. Add 0.8 mL of the prepared NaOH solution to the beaker to raise the pH of the solution to above pH 7 (see Note 5).

8. Weigh out 50 mg (23.8 μmoles) PEG, transfer to a 2 mL micro-centrifuge tube, add 1 mL of MES buffer, and then vortex for 2 min until the solution becomes clear.

9. Add the PEG solution to the beaker dropwise and stir for 24 h.

10. If precipitates are noticed, filter the beaker contents using 0.22 μm syringe filter and then dialyze against 2 L of nanopure water using 1 kDa MWCO dialysis membrane, and change dialysis water at 2, 4, 6, and 24 h, with a total period of dialysis of 48 h.

11. Transfer the dialysis bag contents to a 50-mL conical tube, freeze at −80°C for 30 min, and then lyophilize for two days.

12. Weigh out the dried powder of PEG-Gal to calculate the yield, and then protect from moisture and keep at −20°C for the following conjugation step (we usually collect approximately 40 mg, and the later calculations are based on that yield).

3.1.3. Conjugation of the PEG-Gal free carboxylic group to the PAMAM amine group to form PAMAM-PEG-Gal.

In this second step, the free carboxylic group of PEG in the PEG-Gal conjugate will be activated with EDC/sulfo-NHS and then the activated PEG-Gal-NHS will form an amide bond with free surface amine groups of PAMAM.

1. Remove PAMAM, PEG-Gal, EDC, sulfo-NHS, and MES buffer from the fridge/freezer and let them reach room temperature before use.

2. Add 20 mL of MES buffer to the 50-mL tube containing the PEG-Gal (40 mg = 16.4 μmoles), vortex until the solution becomes clear, transfer to the 30-mL glass beaker, and start stirring.

3. Weigh out 31.44 mg EDC (164 μmoles) and 89.04 mg sulfo-NHS (410 μmoles), transfer both to the same 2 mL micro-centrifuge tube, add 1 mL of MES buffer and then vortex for 2 min until the solution becomes clear (see Note 6).

4. Add the EDC and sulfo-NHS solution to the beaker containing PEG-Gal solution dropwise while stirring, in order to form the NHS ester of PEG-Gal (PEG-Gal-NHS).

5. Stir the solution for 15 min (see Note 7).

6. Add 0.8 mL of prepared NaOH solution to the beaker to raise the pH of the solution above pH 7.

7. Add 1157 μL of G5 PAMAM solution to the beaker drop-wise and stir for 24 h (see Note 8).

8. If precipitates are noticed, filter the beaker contents using a 0.22 μm syringe filter and then dialyze against 2 L of nanopure water using 10 kDa MWCO dialysis membrane, and change dialysis water at 2, 4, 6, and 24 h, with a total period of dialysis of 48 h.

9. Transfer the dialysis bag content to a 50-mL conical tube, freeze at −80°C for 30 min, and lyophilize for two days.

10. Weigh out the dried powder of PAMAM-PEG-Gal to calculate the yield, and then protect from moisture and keep at −20°C until ready for the following steps.

3.2. Characterization of PAMAM-PEG-Gal:

3.2.1. ¹H NMR spectroscopy:

This experiment is carried out to confirm successful synthesis and to elucidate the composition of the final conjugate.

1. In order to confirm Gal conjugation to PEG, dissolve Gal, PEG, PEG-Gal in deuterated chloroform at concentrations of 10 mg/mL to check the conjugation ratio between PEG and Gal (see Note 9).

2. In order to confirm PEG conjugation to PAMAM, and quantify the amount of conjugated PEG, dissolve PAMAM, PEG-Gal and PAMAM-PEG-Gal in deuterated water at 10 mg/mL (see Note 10).

3.2.2. Mass Spectrometry:

This experiment is carried out to confirm the presence of Gal in the final conjugate of PAMAM-PEG-Gal.

1. Dissolve Gal, PEG-Gal, PAMAM-PEG-Gal in water at two different concentrations: 10 and 1 μg/mL and look for the Gal peak using negative electrospray ionization scanning mode.

3.3. Preparation of Nanoplexes of PAMAM-PEG-Gal and pDNA:

Nanoplexes are prepared at different nitrogen to phosphate (N/P) ratios. Nitrogen represents the free surface amine groups on the positively charged polymer, PAMAM. Phosphate represents free phosphate groups on the genetic material, pDNA.

3.3.1. Preparation of stock solutions.

1. Prepare 1mL of PAMAM-PEG-Gal stock in DNAse RNAse free water at 5 mg/mL.

2. Prepare 4 mL of pDNA stock in DNAse RNAse free water at 0.1 mg/mL.

3.3.2. Calculation of N/P ratios.

The following equation is used to calculate the required amounts of PAMAM-PEG-Gal and pDNA needed to prepare nanoplexes at a specific N/P ratio.

$$\frac{\text{N}}{\text{P}}=\frac{\text{Wt}\text{of}\text{PAMAM-PEG-Gal}(\mathrm{\mu }\text{g})/\text{Average}\text{Mw}\text{of}\text{PAMAM-PEG-Gal}\text{that}\text{contains}\text{one}\text{N}\text{atom}(\text{g}/\text{mole})}{\text{Wt}\text{of}\text{pDNA}(\mathrm{\mu }\text{g})/\text{Average}\text{Mw}\text{of}\text{pDNA}\text{that}\text{contains}\text{one}\text{P}\text{atom}(\text{g}/\text{mole})}$$ (Equation 1)

1. From the ¹H NMR data we usually find that the PEG:Gal ratio is 1:1 in the PEG-Gal conjugate and this gives rise to a new Mw of PEG-Gal of 2440.3 g/mole.

2. From the ¹H NMR data, we usually find approximately 6 molecules of PEG-Gal attached to PAMAM, and thus the new Mw of PAMAM-PEG-Gal = 28824.81 (Mw of PAMAM) + (6 × 2440.3g/mole, Mw of 6 PEG-Gal molecules) − (6×18, Mw of 6 water molecules lost during formation of amide bonds) = 43,358.6 g/mole (see Note 11).

3. Since PAMAM G5 has 128 surface amine groups, and since 6 amine groups are occupied with PEG-Gal, this leaves 122 surface amine groups available for complexation with pDNA. Thus, the average Mw of PAMAM-PEG-Gal that contains one N atom will be equal to 43,358.6/122= 355.4 g/mole.

4. The average Mw of pDNA that contains one P atom is calculated as 330 g/mole. This value is based on averaging the Mw of the 4 DNA bases, where an assumption is placed that the 4 bases are uniformly distributed throughout the whole pDNA length (see Note 12).

5. Based on those calculations, Equation 1 can be reduced to the following:

   $$\frac{\text{N}}{\text{P}}=\frac{\text{Wt}\text{of}\text{PAMAM-PEG-Gal}(\mathrm{\mu }\text{g})/355.4(\text{g}/\text{mole})}{\text{Wt}\text{of}\text{pDNA}(\mathrm{\mu }\text{g})/330(\text{g}/\text{mole})}$$ (Equation 2)

6. Based on Equation 2, when starting with 50 μg of pDNA, and wanting an N/P ratio of 1, 54 μg of PAMAM-PEG-Gal will be required, which is equivalent to using 11 μL of PAMAM-PEG-Gal stock (Table 1).

Table 1:

Calculated amounts and volumes of PAMAM-PEG-Gal needed to prepare nanoplexes at different N/P ratios.

N/P ratio	PAMAM-PEG-Gal (μg)	Equivalent volume from 5 mg/mL PAMAM-PEG-Gal stock (μL)
0.5	27	5
1	54	11
5	269	54
10	538	108

3.3.3. Preparation of Nanoplexes.

Nanoplexes are prepared by adding equal volumes of positively charged polymer to that of the negatively charged pDNA solution.

1. For each N/P ratio, prepare two 2-mL microcentrifuge tubes, one labeled as Tube A (+ve) and to this one add 500 μL of the specified concentration of PAMAM-PEG-Gal solution; and the other one labelled as Tube B (−ve), and to this one add 500 μL of the specified concentration of pDNA solution. For example, in order to prepare nanoplexes at N/P of 1, for Tube A add 489 μL of DNAse RNase free water and then add 11 μL of the 5 mg/mL PAMAM-PEG-Gal solution. For Tube B, add 500 μL of 0.1 mg/mL pDNA solution (Table 2).

2. For each specified N/P ratio, transfer the contents of Tube A to that of Tube B and vortex for 30 sec at 1000 rpm, and then leave the nanoplex on the bench for 30 min before performing any further experiments (see Note 13)

Table 2:

Detailed composition of Tube A and Tube B required for the preparation of nanoplexes.

N/P ratio	Tube A (+ve)		Tube B (−ve)
	Volume of 5 mg/mL PAMAM- PEG-Gal (μL)	Volume of DNAse, RNase, free water required to top solution volume to 500 μL (μL)	Volume of 0.1 mg/mL pDNA (μL)
0.5	5	495	500
1	11	489	500
5	54	446	500
10	108	392	500

3.4. Characterization of Nanoplexes of PAMAM-PEG-Gal and pDNA:

3.4.1. Size and Surface Charge Measurements.

Add 100 μL of the prepared nanoplexes to 900 μL of DNAse RNAse free water and measure their size and charge using the Zetasizer Nano ZS.

3.4.2. Electrophoretic mobility.

Gel electrophoresis is carried out to confirm the ability of the nanoplexes to retain pDNA thus preventing migration of the pDNA into the gel. Heparin is added to the nanoplexes to determine the potential of the nanoplexes to release pDNA once taken up by the targeted cells.

1. Add volumes of nanoplexes equivalent to 1 μg pDNA (20 μL in our case) to the wells of a 2% agarose gel stained with 1 μg/mL ethidium bromide.

2. In order to test the ability of heparin to release complexed pDNA from the formed nanoplexes, mix 250 μL of heparin solution at 10 mg/mL with 250 μL of the nanoplex solution, incubate for 15 min at room temperature and then add 40 μL of the mixture to the gel (≅1 μg pDNA).

3. Run the gel for 2 h at 150 Volts.

4. Image the gel using an iBright FL1000 Imaging System or any appropriate imaging system and look for the absence of migrating bands which indicate the formation of stable nanoplexes; then look for the migrating bands with the formulations premixed with heparin. A typical gel image is shown in Figure 1.

Figure 1:

Gel electrophoresis assay showing the ability of nanoplexes, prepared at N/P ratios of 5 and 10, to retain pDNA and therefore inhibit pDNA migration into the gel. Also shown is the ability of the same nanoplexes to release the pDNA once pretreated with heparin.

3.4.3. Surface morphology using TEM.

1. Add one drop of the prepared nanoplexes to the TEM grid for 30 sec, and then remove the excess liquid using a filter paper.

2. If negative staining is required, then to the sample prepared in step 1, add a drop of 1% (w/v) phosphotungstic acid solution for 30 sec, and then remove the excess liquid using a filter paper.

3. Image the prepared samples using TEM. Typical TEM images are shown in Figure 2.

Figure 2:

TEM images of nanoplexes prepared at the N/P ratio of 10, and imaged directly (A & B), or imaged using negative staining (C & D).

4. Notes

1. Nanopure water is used during all the steps of conjugate synthesis, however, DNAse and RNAse free water is used during the preparation and characterization of nanoplexes of PAMAM-PEG-Gal conjugate and pDNA.

2. Some of the chemicals used during this experiment are hygroscopic, and thus need to be handled carefully, and avoid opening their containers until they reach room temperature to avoid any errors during the weighing process.

3. Calculation of Gal, EDC and sulfo-NHS amounts is based on using 50 mg of PEG (Mw 2100, 23.8 μmoles). We use a 10x molar excess of Gal (238 μmoles) in order to assure that all the PEG amine groups are conjugated with Gal. We then use equivalent amounts of EDC to that of Gal (238 μmoles) in order to convert the Gal carboxylic group to an EDC ester, and then 2.5x molar excess of sulfo-NHS as compared to EDC (2.5 × 238 = 595 μmoles) in order to assure that all of the formed unstable Gal-EDC esters are converted to the stable Gal-NHS esters.

4. 2-Mercaptoethanol is added at this step to quench excess EDC, and thus avoid activation of the free PEG carboxylic group once added to the solution, which, if it happens, might result in the formation of amide bonds between PEG molecules, and subsequently reduce the yield of the PEG-Gal. The amount of 2-mercaptoethanol used in this step is based on using a 10x molar excess as compared to that of EDC which is equivalent to using 2380 μmoles of 2-mercaptoethanol. Based on 2-mercaptoethanol Mw and density, the 2380 μmoles is equivalent to 168 μL.

5. NHS esters (Gal-NHS) require slightly alkaline pH in order to form stable amide bonds with compounds bearing free amine groups, which is PEG in this case.

6. Calculations of the amounts of EDC, sulfo-NHS, and PAMAM used in this second step are based on two important factors. (a) The yield of PEG-Gal from the former step, and (b) the fact that only 8% of PAMAM surface amine groups are needed to conjugate with PEG-Gal, as this ratio has been found to mitigate PAMAM toxic effects without affecting transfection efficiency [22].

   1. We usually collect approximately 40 mgs of PEG-Gal (Mw = 2440.3, 1:1 conjugation between PEG and Gal) from the first step, which is equivalent to 16.4 μmoles.

   2. We use 10x molar excess of EDC as compared to PEG-Gal (164 μmoles = 31.44 mg) and 2.5x molar excess of sulfo-NHs as compared to EDC (410 μmoles = 89.04 mg).

   3. PAMAM G5 has 128 surface amine groups, and 8% of that value is 10.24 amine groups, thus conjugating 1 μmole of PAMAM with 10.24 μM of PEG-Gal, will result in reacting only 8% of PAMAM surface amine groups.

   4. Since we have 16.4 μmoles of PEG-Gal, we will need 1.6 μmoles of PAMAM.

   5. Using 1.6 μmoles of PAMAM (Mw 28824.81) is equivalent to using = 46.12 mg.

   6. Since PAMAM is provided in a methanolic solution at 5% w/w, then 46.12 mg will be equivalent to using 922.4 mg of the solution, and based on PAMAM density of 0.797 g/mL, 922.4 mg will be equivalent to 1157 μL.

7. There is no need to add 2-mercaptoethanol at this step, because all of the free carboxylic groups available have already been activated in the solution and the excess EDC will not have any negative effect on the reaction.

8. The stirred solution needs to be protected from light, by transferring the beaker to a dark room, or by wrapping the beaker with foil. Keep in mind that methanol needs to be evaporated overnight from your beaker, so do not cover your beaker tightly.

9. In order to get a clear ¹H NMR spectrum, you will need to sonicate the samples for 10 min after adding deuterated chloroform to maximize their solubility because of their limited chloroform solubility, and then try to increase the number of scans as much as possible when capturing the ¹H NMR spectrum. It is worth mentioning that deuterated water was avoided as a solvent in this step because of the overlap with the distinct Gal peak.

10. In order to estimate the molar ratio of PEG: Gal in the PEG-Gal conjugate, and that of PEG-Gal: PAMAM in the PAMAM-PEG-Gal conjugate, you need to integrate the peaks of their distinct protons in the ¹H NMR spectrum. For the PEG-Gal conjugate dissolved in deuterated chloroform, peaks at 3.5 and 4.5 ppm represents 180 H of PEG (-CH₂CH₂O-) and 1H of Gal, respectively[23,24]. For the PAMAM-PEG-Gal conjugate dissolved in deuterated water, peaks at 2.3 and 3.5 ppm represents 504 H of PAMAM (-CH₂CONH-) and 180 H of PEG-Gal (-CH₂CH₂O-), respectively [25].

11. Please note that the original calculations of the PEG:PAMAM ratio are based on conjugating 8% of surface amine groups of PAMAM with PEG (see Note 6), which is equivalent to 10.24 molecules of PEG to one PAMAM molecule. However, our ¹H NMR data shows that we were successful only in conjugating 6 PEG molecules to each PAMAM molecule, and that is why the calculations of the N/P ratio are based on 6 PEG molecules and not 10.24 molecules.

12. If the exact sequence of pDNA is known, more accurate calculations of pDNA average Mw can be estimated. In addition, if RNA-based genetic material is used, then the average Mw will be lower (325 g/mole) because of the lower Mw of uracil in the RNA as compared to that of thymine in the pDNA. It is worth mentioning that designing siRNA delivery systems is more relevant in NASH therapy [1]. However, in our protocol we decided to use pDNA, because we were interested in developing a universal gene delivery system that works well with any type of genetic material. It is usually harder to deliver pDNA when compared to the smaller-sized siRNA, and thus successful delivery of pDNA indicates success with siRNA, but not the opposite.

13. Vortexing at a higher speed or using microcentrifuge tubes of different sizes might result in the aggregation of the formed nanoplexes. Thus, we recommend adhering to the above-mentioned protocol.