Preparation and Characterization of Engineered VEGF-C
Preparation and characterization of reverse micelles (RMs)
A high-pressure homogenization/microfluidization technique previously reported by Bai et al. (2019) was followed with a slight modification for the preparation of RMs. To prepare RMs, Distearoyl-rac-glycerol-PEG2K (DSG-PEG 2000) and Span 80 were dissolved in an organic solvent (ethanol). Span 80 was used as the emulsifier. Double-distilled water containing VEGF-C was added dropwise to the organic phase and mixed well with a digital magnetic stirrer (IKA®-Werke GmbH & Co. KG, Stäüfen, Germany). The coarse dispersion was then passed through a benchtop high-shear homogenizer (LM20, Microfluidics, Massachusetts, USA) at 20,000 pressure for three cycles to obtain nanosized translucent RMs (Supp Fig. 1).
Mean particle size (MPS), polydispersity index (PDI) and zeta potential
The MPS, PDI, and ZP values were evaluated using dynamic light scattering (DLS). One hundred microliters of RM sample were diluted with double distilled water up to 1 ml, in triplicate. Using a Malvern Zetasizer (Nano ZS, Malvern Instrument Ltd, Malvern, UK), the MPS, PDI, and ZP values of the diluted RMs were determined at 25°C by placing 1 ml of sample directly into a standard quartz cuvette. A He-Ne light source at 633-nm wavelength was used as the source of the laser beam in this instrument.
Stability study
the RM formulation was observed over a period of one month at 4°C for the stability study. Samples were withdrawn at regular time intervals of 0, 1, 2, 3, and 4 weeks to observe the influence of storage conditions on MPS, PDI, and ZP values.
pH measurement
The pH of the RM formulation was measured in triplicate using a pH meter (Mettler Toledo, Greifensee, Switzerland) at room temperature.
Field emission-scanning electron microscope (FE-SEM)
The surface morphology and size of the RM formulations were examined using FE-SEM (GeminiSEM 500, Carl Zeiss Microscopy, GmbH, Oberkochen, Germany). Before loading the sample into the FE-SEM instrument, it was coated with gold to avoid or minimize the charging effect. Images were recorded at a voltage–2–4 kV using an in-lens detector [44].
Atomic force microscopy (AFM)
AFM (Innova SPM, Bruker, Germany) analysis was performed in the non-contact tapping mode to record the topographical images of the optimized RM formulation. The samples were prepared using the drop-casting method, followed by drying at room temperature. The raw data obtained from the system were processed using Gwyddion software, version 2.60.
Field emission-transmission electron microscopy (FE-TEM)
The morphologies and sizes of the RMs were examined using FE-TEM (JEOL, 2100F, Japan). Approximately 10 µl of the sample was dropped onto a carbon-coated copper grid and covered with collodion carbon, followed by air-drying at room temperature before measurements were taken. Images were recorded at an accelerating voltage of 90 kV, and images were recorded.
Determination of encapsulation efficiency (EE %)
The percentage of VEGF-C encapsulated in the formulation was quantified by enzyme-linked immunosorbent assay (ELISA) using the Quantikine™ human VEGF-C ELISA kit protocol [45]. The prepared reverse micelles were placed in a rotary evaporator (IKA®, Werke GmbH & Co. KG, Stäüfen, Germany) to remove the solvent present in the formulation at 58º C temperature, 100 rpm, and − 700 to -800 mbar pressure. The thin film obtained was hydrated using double-distilled water. One milliliter of the redispersed formulation was taken and 1 ml of ethanol was added to disrupt the self-assembled structure of the lipid layer. The samples collected in triplicate were analyzed using an ELISA kit at 450 nm with a multimode reader (SpectraMax®, CA, USA) according to the manufacturer’s instructions. The EE % was calculated using Eq. (1).
$$\text{E}\text{E} \text{\%}=\frac{\text{A}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} VEGF-C \text{i}\text{n} \text{f}\text{o}\text{r}\text{m}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}}{\text{A}\text{c}\text{t}\text{u}\text{a}\text{l} \text{a}\text{m}\text{o}\text{u}\text{n}\text{t} \text{o}\text{f} VEGF-C\text{a}\text{d}\text{d}\text{e}\text{d} \text{i}\text{n} \text{f}\text{o}\text{r}\text{m}\text{u}\text{l}\text{a}\text{t}\text{i}\text{o}\text{n}}\times 100\dots .\text{E}\text{q}.\left(1\right)$$
In vitro VEGF-C release from reverse micelles
The in vitro VEGF-C release from the RMs was analyzed by ELISA using the Quantikine™ human VEGF-C ELISA kit protocol [46]. Two milliliters of reverse micelle dispersions containing 666.6 ng VEGF-C were placed in the middle of the dialysis bag. The dialysis bag containing the dispersions was then kept in 5 ml of release medium (phosphate buffer saline, PBS, pH 7.4) in a 50 ml falcon tube. The Falcon tube was then placed in an incubator shaker (REMI Sales & Engineering Ltd., Mumbai, India) at 37°C with shaking at 75 rpm. The samples were withdrawn at different time points (1, 2, 4, 6, 8, 10, and 24 h), and pre-warmed fresh medium was added at each time point. The collected samples were stored at -20°C for later analysis. The collected samples were analyzed according to the manufacturer’s instructions in the ELISA kit booklet using an ELISA kit at 450 nm with a multimode reader (SpectraMax®, CA, USA). The results are expressed as percent cumulative release ± standard deviation (SD) in triplicate.
The percent cumulative release data were fitted into different release kinetic models, such as zero-order, first-order, Korsmeyer-Peppas, Weibull, Higuchi, and Hixson-Crowell models, using KinetDS software, version 3.0. The best-fit plot was chosen based on the maximum R2 (coefficient of determination) value [47].
In vivo studies
Study Groups and treatment
All animals received humane care according to the criteria outlined in the ‘‘Guide for the Care and Use of Laboratory Animals’’ prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86 − 23 revised 1985). The study was approved by the Animal Ethics Committee of ILBS, New Delhi, according to standard guidelines (Ethics Protocol No: IAEC/ILBS/18/01). Rats were housed in a room at 22 ± 30C a 12 hrs. light-dark cycle and were given food and water ad libitum. Studies were performed on thirty-six 8-week-old male Wistar rats and twelve male 8–10-week-old Sprague Dawley (SD) rats weighing 250–300 g. Wistar rats were used to develop cirrhotic models of portal hypertension, while SD rats were utilized for the development of non-cirrhotic portal hypertension models by partial portal vein ligation (PPVL). Animals were randomized into three groups for the cirrhosis study: healthy control group (treated with saline), CCl4-V or vehicle group (CCl4 plus lipid nanocarriers alone without VEGF-C), and E-VEGF-C group (CCl4 plus E-VEGF-C). Cirrhosis was induced by intraperitoneal injection of 1.0 ml/kg body weight of CCl4:olive oil in 1:1 ratio, two times a week for 12–14 weeks until ascites formation. The control group (control) received olive oil 1.0 ml/kg intraperitoneal injection (i.p.) two times a week for 12 weeks and then saline 1.0 ml/kg per orally on alternate days for two weeks during the 13th -14th week. One week after ascites formation without CCl4, lipid nanocarrier formulation (without VEGF-C) was administered via oral route on alternate days for 2 weeks during the 13th -14th week (vehicle). The E-VEGF-C group, along with CCl4, received E-VEGF-C through the oral route at a dose of 600 µg/kg of the body weight on alternate days for 2 weeks during the 13th -14th week. Ascites was graded as mild, moderate, or severe based on fluid volume. Less than 10 ml was mild, 10–30 ml was moderate, and > 30 ml was severe. These rats were sacrificed 48 hrs. after the last dose of saline, vehicle, or E-VEGF-C, after performing the hemodynamic studies.
The two groups were prepared for the PPVL study. PPVL vehicle (PPVL with nanocarriers alone) and PPVL + E-VEGF-C. Non-cirrhotic animal models of portal hypertension were developed using partial portal vein ligation (PPVL), as previously described [48]. Briefly, under isoflurane anesthesia, using a single ligature of 3 − 0 silk tied around the portal vein, a calibrated constriction was performed with a 19-gauge blunt-tipped needle. The needle was then removed, leaving a calibrated constriction of the portal vein. For the PPVL animals, the two groups were randomized. In the PPVL + vehicle group, the lipid nanocarrier formulation was administered orally on alternate days for 2 week after the PPVL surgery. In the PPVL + E-VEGF-C group, E-VEGF-C was orally administered at a dose of 600 µg/kg of the body weight of the animal on alternate days for 2 weeks one day after the PPVL surgery week. In a few rats, the plasma volume was measured using Evans Blue dye.
In vivo biodistribution and half-life studies
E-VEGF-C (single dose: 300µg/kg) tagged with coumarin-6 dye was administered on day 0 in healthy animals and CCL4 rats after 12 weeks. Rats were euthanized 2 hrs. after a single oral administration of E-VEGF-C for the collection of liver, intestine, mesentery, spleen, lung, and kidney tissues. The tissues were weighed and homogenized with physiological saline, and fluorescence intensity was detected at 520 nm using a spectrofluorometer. Some parts of the tissues were also observed under a fluorescence microscope for visual detection of labeled nanoparticles. The fluorescence intensity values of coumarin were normalized to those of the untreated control rats. Levels of VEGF-C were determined per mg protein of the tissues using the human VEGF-C ELISA kit (Elabsciences, Houston, USA) according to the manufacturer’s protocol.
For plasma half-life studies, after a single oral injection of E-VEGF-C, serial blood samples (300 µl) were collected from the rats in EDTA vials at 10 min, 20 min, 30 min, 1 hr, 5 hr, 10 hr, and 24 hr. After 20 min of incubation at room temperature, blood samples were centrifuged at 1200× g for 10 min. The plasma (100–150 µl) was collected, snap frozen, and stored at − 80°C. The amount of VEGF-C in each sample was determined using a human VEGF-C ELISA kit, as previously described. Plasma VEGF-C concentration–time data were analyzed at different time points.
Analysis of Plasma Volume
Plasma volume was measured using Evans blue dye as previously described [12]. Briefly, 0.2 ml of Evans Blue (Himedia labs) solution (3 mg) was injected through a jugular vein catheter, followed by saline to clear the dye from the catheter. After five, 1 ml of blood was withdrawn from the femoral artery catheter. The plasma aliquot was diluted ten times in distilled water, and the absorbance was read at 600-nm using a spectrophotometer. Plasma volume (ml) was calculated using the following formula:
Plasma volume (ml) = Absorbance of standard/Absorbance of sample × 10, where the standard was 3 mg of Evans blue dye in 10 ml of plasma diluted by a factor of 10
Analysis of Lymphatic Transport using Tracer dye BODIPY FL-C16
To evaluate lymphatic drainage in the mLVs, a long-chain fluorescent fatty acid (BODIPY, Thermo fisher), known as lymphatic tracer, was administered orally with olive oil in 1:10 ratio with final volume of 200ul. After 2 hr, mesentery tissue was isolated and optically cleared using methyl salicylate. Images of mesenteric lymphatic drainage were obtained using confocal microscopy at 10X (Leica SP8). Six fields from each slide were randomly selected and photographs were taken. Lymphatic drainage was quantified by measuring BODIPY fluorescence intensity inside the LVs using ImageJ. LVs leakage was quantified by measuring BODIPY fluorescence intensity in vicinity to LVsusing ImageJ. Diameter of the mLVs were calculated using ImageJ diameter plugin.
Assessment of Hepatic and Systemic Hemodynamic Parameters
Rats were anesthetized with ketamine hydrochloride (60 mg/kg) and midazolam (3mg/kg) intraperitoneally, fastened to a surgical board, and maintained a constant temperature of 37°C ± 0.5°C. Tracheostomy and endotracheal cannulation (PE-240 catheter; Portex, Minneapolis, MN, USA) were performed to maintain adequate respiration during anesthesia. The femoral artery and ileocolic vein were cannulated with PE-50 catheters to measure mean arterial pressure (mmHg) and portal pressure (PP) (mmHg), respectively. A non-constrictive perivascular ultrasonic transit time flow probe (2PR, 2-mm diameter; Transonic Systems Inc., Ithaca, NY, USA) was placed around the portal vein as close as possible to the liver to measure portal blood flow (PBF) (mL/min). Intrahepatic vascular resistance (IHR) (mmHg ·mL·min–1·g–1) was calculated as PP/PBF. Blood pressure and flow were registered on a multichannel, computer-based recorder using Chart, version 5.0.1, for Windows software (PowerLab; AD Instruments). Hemodynamic data were collected after a 20-minute stabilization period
Computed tomography analysis
Computed tomography (CT) was performed 48 h after treatment to visualize ascites. The animals were evaluated using Somatom Definition AS plus 128 Acquisition 384 slice reconstruction (Siemens Healthineers, Forchheim, Germany).
In vitro Studies
Isolation of Lymphatic endothelial cells from mesenteric tissue of rat and uptake of E-VEGF-C in vitro
For the isolation of mesenteric LyECs, protocol was adapted from Ribera et al., 2013 with minor moddifaction. Briefly, the rats were anesthetized with ketamine hydrochloride (60 mg/kg) and midazolam (3mg/kg) intraperitoneally. A midline incision was made to perfuse the mesentery with saline by inserting the catheter into the portal vein. Clear mesenteric tissue was extracted and placed in DMEM supplemented with 2% antibiotic and antimycotic solutions. Sterile scissors were used to finely mince the tissue into ~ 1 mm3 pieces. The minced tissue was centrifuged at 2500 rpm for 10 min at RT and washed twice with PBS. For in vitro digestion, 0.25% collagenase IV was previously prepared and prewarmed at 37 C, tissue was resuspended in enzyme solution supplemented with 3mM CaCl2 at constant shaking for proper digestion. Following digestion for 30 min at 37 C, the tube was transferred to a biosafety cabinet, passed the digested tissue suspension through a 70 µm strainer placed into a sterile 50 ml tube. Equal amounts of sterile DMEM were passed through a strainer to inactivate the enzyme. The cell suspension was then centrifuged at 1200 rpm for 5 min at 4°C and washed twice with PBS.
For sorting of LyECs, LyEC-specific primary antibody podoplanin (pdpn, 1:200) and CD31 (1:100) was added in the cell suspension with CD45 and 7AAD, and cells were incubated on ice for 30 mins. After washing, secondary antibody conjugated with fluorochrome was added at a 1:500 dilution and incubated on ice for 30 min. Thereafter, sorting was performed under sterile conditions using a BD FACS Aria, and the cells were collected in EGM-2 media. Collected cells were washed with PBS, resuspended in EGM-2 medium, and seeded on pre-coated fibronectin plates.
The cultured cells were incubated with nanoengineered VEGF-C particles for 30 min at 37 C in 5% CO2. After incubation, the cells were rinsed with PBS to remove the remaining nanoparticles. Cells were then fixed for 7 min at room temperature in the dark using a permeabilization and fixing kit (BD Cytofix/Cytoperm) and washed once with BD perm/wash buffer[29]. Staining was performed with DAPI (4,6-diamidino-2-phenylindole) at a 1:2000 dilution. Fluorescence of coumarin-6 labeled VEGF-C particle and DAPI-stained cells were imaged using an inverted fluorescence microscope (Evos microscope) in the green and blue channels, respectively. Fluorescence was quantified using the ImageJ software.
Assessment of blood and lymphatic vessels by immunohistochemistry and immunofluorescence
Tissues samples were fixed in 10% buffered formalin and processed. Sections of 7-µm-thick paraffin-embedded tissues were heat fixed and deparaffinized at 45 C and rehydrated in a descending ethanol series. Following antigen retrieval by heating for 8 minutes in a microwave with citrate buffer, sections were incubated for 20 minutes with peroxidase 1 solution to quench endogenous peroxidase. Protein blocking was done 3% BSA. Tissue slides were then incubated overnight at 4C in a humid chamber with anti-podoplanin mAb, and staining was completed using the HRP-conjugated mouse/rar/human detection kit and DAB chromogen as a substrate, according to the manufacturer’s instructions. Last, sections were counterstained with Hematoxylin and eosin for 1 minutes. The slides were mounted with a coverslip using Mounting Media. In immunofluorescence, after primary antibody incubation, secondary antibody attached to fluorochrome is added for 1 hr at RT and slides were mounted with Vectashield mounting media with DAPI. Six fields from each slide were randomly selected, and photographs were taken using an inverted fluorescent microscope (Nikon Instruments, Inc.) and quantified using ImageJ software. Details of the antibodies used are provided in S Table 4.
Assessment of Bacterial Translocation
GFP labelled salmonella typhimurium were cultured in Luria Broth with ampicillin and 109 cells were orally gavaged in rats for 48 hrs. All experiments were performed under sterile conditions. Rats were Anesthetized with ketamine hydrochloride (60 mg/kg) and midazolam (3mg/kg) intraperitoneally, shaved, and the skin was disinfected with alcohol. Subsequently, after midline laparotomy, MLNs were dissected, removed, and weighed in sterile condition. Tissue samples of liver, spleen, and lung were also removed and weighed. All specimens were diluted in phosphate-buffered saline (100uL per 100mg) and homogenized, and suspension was cultured on Luria broth agar with ampicillin and observed after 24 hrs. Presence of GFP positive bacteria using UV transilluminator was considered evidence of BT to different organs. To test the translocation of bacteria in blood, 5 ml of blood was also collected in sterile condition and added to blood culture bottle and incubated at 37 C. After 1 hr. of incubation, 100 ul of blood were collected from culture bottle and spread on LB agar plate and incubated at 37 C.
Immune cell quantification using Flow Cytometry
Cells were isolated from Mesenteric lymph node using enzymatic digestion by collagenase type IV at 37 C for 10 mins and single cell suspension was prepared using 40-micron sterile filter. Rest of the tissue was was passed through the filter using 5 ml syringe plunger. Single cell suspension was washed with PBS and counted. Cells from blood was isolated using RBC lysis buffer. 9 ml of RBC lysis buffer 1X was added to 1 ml of blood and centrifuged at 1500 rpm, 25 C for 5 min. cells were washed with PBS and counted. Half million cells were incubated with antibodies specific for T cell subsets and dendritic cells and incubated for 30 min to 1 hr in dark at 4C. 1 lakh events were acquired for each experiment.
Detection of TNF-α and endotoxins in the Ascitic fluid and Systemic Circulation
TNF-α levels were assessed using a TNF-α ELISA kit (Thermo Fisher Scientific, Massachusetts, USA) as recommended by the manufacturer’s protocol. Endotoxin levels in the serum were assessed using a chromogenic kinetic limulus ameobocyte lysate assay kit, following the manufacturer's instructions (Thermo Fisher Scientific, Massachusetts, USA).
RNA extraction and RT-PCR
RNA extraction and RT-PCR were performed on excised mesenteric tissues stored in an RNA buffer. Total RNA was isolated using a Nucleopore kit, according to the manufacturer’s instructions. RNA was quantified at 260/280 nm using a Nanodrop 2000 spectrophotometer (Thermo Scientific). First-strand cDNA was synthesized from 1µg of total RNA using reverse transcriptase (Thermo Fisher Scientific Verso cDNA synthesis kit) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using SYBR green PCR master mix (Fermentas Life Sciences) on a ViiA7 instrument PCR system (Applied Biosystems, USA). The following cycling parameters were used: start at 95°C for 5 min, denaturation at 95°C for 30 s, annealing at 60°C for 30 s, elongation at 72°C for 30 s, and a final 5 min extra extension at the end of the reaction to ensure that all amplicons were completely extended and repeated for 40 amplification cycles. Relative quantification of the expression of relevant genes was performed using the ΔΔCt method after normalization to the expression of the housekeeping gene GAPDH. Primer sequences used are listed in S Table S5.
Western Blotting
Mesenteric tissues were crushed in liquid nitrogen, and 100 mg of tissue powder was added to 200 µL of RIPA lysis buffer (Merck, sigma 20–188). Homogenization was performed on ice until a clear solution was obtained. After centrifugation at 12,000 rpm for 20 min, the supernatant was collected in fresh microcentrifuge tubes and incubated on ice for 30 min. The protein content of the tissue lysate was measured using a BCA kit (Thermo Fisher Scientific, Waltham, MA, USA). Protein samples were denatured at 95°C for 5’ in Laemmli buffer. 60 µg of protein was loaded into each well and separated by 10% SDS-PAGE. The gel was run at 80 V for approximately 2 hr. Proteins were electroblotted onto activated PVDF membrane at 60 V for 2 hr. at 4°C, and the membrane was blocked in 5% BSA in Tris-buffered saline containing 0.05% Tween for 2 hr. Membranes were blotted with various primary antibodies i.e., VEGF-C and GAPDH overnight at 4°C, followed by the appropriate HRP-conjugated anti-rabbit and anti-mouse secondary antibodies for 2 hr. The membrane was then treated with the chemiluminescence ECL, and visualized on gel doc (Invitrogen, iBrightCL1500). Densitometry was performed using NIH software (ImageJ). Details of the antibodies used are provided in S Table 4.
Statistical Analysis
Continuous variables are expressed as either mean ± standard deviation for continuous distribution or as median values for skewed distribution. Continuous variables were compared between the two groups using an unpaired two-tailed Student’s t-test or Mann-Whitney U test. Variables greater than 2 were compared using one-way ANOVA followed by a post hoc Tukey test. Bar diagrams with various data points, dot plots, and box whisker plots were plotted using GraphPad Prism (version 8.0.1.; GraphPad Software, San Diego, CA, USA), and statistical analysis was performed using GraphPad Prism. Statistical significance was set at p < 0.05.