Abstract
Nanoparticles are becoming increasingly used in medicine as carriers of anti-tumor drugs.
Published evidence suggests that they produce better patient outcomes than current delivery systems. I
have investigated a little studied nanoparticle coating, tetra-ethylene glycol (TEG), to determine if it
provides pharmacologically relevant advantages, such as increased serum half-life and resistance to
protein adsorption. I compared the TEG-coated particles to those coated with glutathione (GSH), which
are relatively unaltered in circulation and readily filtered by the kidney. Nanoparticles coated with GSH
and TEG were intravenously injected into mice and their half-lives and final destinations were determined
via photometric analysis, light microscopy, and inductively coupled plasma mass spectrometry. Protein
adsorption, which facilitates macrophage phagocytosis of nanoparticles, was studied via column
chromatography and gel electrophoresis. I found that the GSH particles had a shorter half-life than the
TEG particles (approximately 10 minutes versus 70 minutes) and predominantly accumulated in the
kidney. The TEG particles predominantly accumulated in the spleen and liver, although kidney proximal
convoluted tubule cells also took them up. The TEG particles were largely unaggregated in serum.
Those aggregates that did form, possibly by transient protein adsorption following ligand exchange
reactions, were taken up by the spleen as shown by light microscopy. These results demonstrate that TEG
nanoparticles have an increased serum half-life and resistance to protein adsorption suggesting TEG could
be an alternative to currently used coating agents. TEG could prove a more efficient coat considering its
minimal impact on nanoparticle size and its synthesis producing greatly mono-dispersed samples.
1. Introduction
Nanoparticles are metal clusters with a diameter of less than 100 nm.1 These materials have the
potential to deliver drugs in a more controlled manner than current solvent-based medical delivery
techniques with Taxol-bound nanoparticles delivering anti-tumor drugs more specifically to oncogenic
cells, limiting side-effects.1,2 I investigated the mammalian processing of gold nanoparticles, which if
better understood could lead to advances of the technology for clinical purposes. Gold was used because
its cellular uptake following introduction to serum is generally not associated with cytotoxicity. 3 Gold
nanoparticles are also readily coated with thiol-containing compounds, such as poly-ethylene glycol
(PEG), that can modulate their pharmacological properties1,3; pre-existing data suggests that nanoparticle
coating impacts their removal from circulation with particles coated with PEG remaining in serum for
hours, minimally interacting with proteins, and causing minor physiological consequences in the organs
that filter them (the liver and spleen via metallophilic macrophages and the kidney via filtration through
the glomerular basement membrane).3,4,5,6 Extensive work has been done studying the in vivo behavior of
these PEG nanoparticles; however, a number of coats have yet to be investigated in live models,
particularly tetra-ethylene glycol (TEG).1 Only four papers could be found documenting use of this
compound as a coating agent, none of which described their in-vivo behavior (it was noted that TEG
conferred water solubility and resistance to nonspecific protein adsorption, both of which are
pharmacologically important).7,8,9,10 In this work, I determined the serum half-life, renal/splenic/hepatic
filtration, and in-vivo reactivity of TEG particles, comparing their behavior to well-documented
glutathione (GSH)-coated particles (quickly excreted and fairly unreactive nanoparticles) to investigate
their potential as a gold nanoparticle coating agent.11
As TEG is chemically similar to the well
documented PEG, I hypothesized that this coating would confer comparable properties pertaining to
increased half-life, non-reactivity, and in-vivo stability.1,9,10
2. Methods
2.1: Chemicals. Bovine serum albumin (BSA), reduced glutathione, and tetra-ethylene glycol were
obtained from Sigma Aldrich (product codes A-3311,G4251, and 767751, respectively).
2.2.1: TEGylated gold nanoparticle synthesis. All aspects of this synthesis were conducted at room
temperature while stirring. A glass bottle had (a) 18 mL of distilled water, (b) 800 µL of 0.1 molal Boric
Acid, (c) 40 µL of 5 M TEG-SH (10 mM final concentration), and (d) 800 µL of 25 mM HAuCl4 added.
The solution was left for one minute and (e) 160 µL of 0.5 molal NaBH4 was then added. The mixture
was left overnight and concentrated in 4 mL Amicon 30 kD filters. All samples were stored in a
refrigerator at 4ºC.
2
2.2.2: GSH-coated gold nanoparticle synthesis. All aspects of this synthesis were conducted at room
temperature while stirring. A glass bottle had (a) 44.3 mL of distilled water, (b) 2 mL of 0.1 molal Boric
Acid, (c) 1 mL of 0.5 molal dithiothreitol (10 mM final concentration), and (d) 2 mL 25 mM HAuCl4
added. The solution was left for ten minutes upon which (e) 400 µL of 0.5 molal NaBH4 was added.
This was left for thirty minutes and (f) 5 mL of 0.5 molal GSH dissolved in 0.5 molal Na 2CO3 was added
(50 mM final concentration). The bottle was left for ten minutes following which its contents were
concentrated in a 70 mL Amicon 30 kD filter. All samples were stored in a refrigerator at 4ºC.
2.3.1: Gold nanoparticle optical density measurement and dilution. Following synthesis, a volume of
each batch of nanoparticles was diluted 1:1000 and analyzed spectrophotometrically at a wavelength of
260 nm. The stock solution of concentrated nanoparticles was subsequently diluted to an absorbance of
0.25 based on the original absorbance:
M1V1 = M2V2 ; A = εbM → A α M ; A1V1 = A2V2 → V2 = A1V1 / A2.
2.3.2: Gel electrophoretic assessment of gold nanoparticle size distribution. Each batch of nanoparticles
was analyzed using gel electrophoresis. A mixture of 10 µL LG buffer (200 μL 80% glycerol with 80 μL
0.5 molal NaHCO3 and bromophenol blue), 1 µL nanoparticles, and 1.2 µL of 10x PBS (~1x final
concentration) was created. This mixture was loaded into a 4-20% Tris/Glycine gel in a solution of
1x Tris/Glycine buffer without sodium-dodecyl sulfate (SDS), subjected to a current, and the unstained
gel was scanned with an Epson Scanner.
2.3.3: Gold nanoparticle core diameter determination. Transmission electron microscopy (TEM) was
used to assess gold nanoparticle core diameter. Dilute particles (1:250 dilution) were placed on a glow
discharged electron microscope grid and left to dry. The core diameters of at least 1000 nanoparticles
were measured using TEM to calculate an average particle core diameter using the program ImageJ
(software available at http://rsb.info.nih.gov/ij/ from the National Institutes of Health).
2.3.4: Gold nanoparticle hydrodynamic radius determination. Column size-exclusion chromatography
was used to assess the hydrodynamic radius of the gold nanoparticles by comparing nanoparticle elution
fraction to the elution fraction of several standards, including IgG, bovine serum albumin, and ovalbumin.
3
2.4: Injection of gold nanoparticles and blood collection from mice. Female B6 mice (B6 Jax), some
splenectomized and permitted to heal for a week, were obtained, weighed, and fed normal diets. (a) A
blood sample was collected from an animal’s retro-orbital sinus in one eye using a heparinized
microcapillary tube.
(b) A solution of 90 μL gold nanoparticles and 10 μL 10x PBS (1x final
concentration) was subsequently created and injected into the animal’s other retro-orbital sinus (controls
were injected with 100 µL of 1x PBS).12 (c) For TEGylated particle injections and associated controls,
blood was collected at 5 min, 166 min, 333 min, and 500 min. For GSH-coated particle injections and
associated controls, blood was collected at 3 min, 10 min, 15 min, and 20 min. (d) At the end of each
experiment, animals were sacrificed via cervical dislocation. (e) The animal’s kidney, spleen, and liver
were subsequently collected and stored in Karnovsky’s Fixative for light microscopic analysis. (f) All
experiments were repeated using a different batch of particles with at least two animals for each
condition.
All invasive procedures were performed following Isoflurane (inhaled anesthesia)
administration and were approved by an IACUC committee.
2.5: Half-life determination of gold nanoparticles. The micro-capillary tubes used to collect blood
samples were centrifuged and scanned on an Epson Scanner set to positive Film mode with a DPI of 1000
and non-reset settings. ImageJ was used to calculate the minimum gray value of the plasma at the center
of each scanned tube, which was inputted into Microsoft Excel. A relative concentration was calculated
for each sample using a best-fit standard curve produced from serial dilutions of undiluted nanoparticles
(1:15, 1:20, 1:25, 1:30, 1:40, 1:60, 1:100, and 1:250 dilutions) (Figure 1). This measurement was
adjusted based on the calculated concentration of the sample collected before sample injection via
subtraction of this background measurement (Figure 1). This data was plotted against time and the
length of the pharmacokinetic alpha phase (the approximate length of time for the material to distribute
throughout the body and penetrate tissues) was calculated by estimating the point at which this curve
began to be roughly linear using a logarithm-based trend line.13 The half-life was determined by plotting
data collected after the estimated alpha phase and solving for the time when particle relative concentration
equated with half of the logarithmic best fit line’s y-intercept.13
4
(a)
(b)
Figure 1. Logarithmic standard curves of nanoparticle serial dilutions. (a) GSH nanoparticle standard
curve. (b) TEG nanoparticle standard curve.
2.6: Gel electrophoresis analysis of plasma samples. Plasma was isolated from the collected blood
samples following centrifugation of the micro-capillary tubes. A volume of 6 µL of sample was then
mixed with 4.8 µL of LG buffer and 1.2 µL 10x PBS (1x final concentration). This mixture was placed
into a Tris/Glycine 4-20% gradient gel without SDS and subjected to a current to observe electrophoretic
migration and band intensity variation relative to particle time in circulation. This gel was scanned using
an Epson scanner.
2.7.1: Tissue sectioning. Kidneys, spleens, and livers were embedded in LR white resin, had sections
taken from abnormally dark portions of the tissues (which signified nanoparticle uptake) if present, and
placed on slides with unstained and toluidine blue stained sections. At least two slides were made from
each experimental condition (both batches of TEGylated particles injected into non-splenectomized
animals, both batches of TEGylated particles injected into splenectomized animals, both batches of GSHcoated particles injected into non-splenectomized animals, both batches of GSH-coated particles injected
into splenectomized animals, and animals injected with saline acting as controls).
2.7.2: Light microscopy analysis.
Slides were analyzed using bright-field light microscopy at
magnifications of 100x and 400x. Particles were identified by dark punctuate that was not visualized on
control sections (not shown). Histological structures were identified using toluidine blue stained sections
5
(Figure 2). Images were captured using the program Volocity, which is available from Perkin Elmer.
Images had their brightness adjusted using ImageJ to facilitate nanoparticle identification.
(a)
(b)
Figure 2. Labeled histological diagram of (a) kidney with proximal convoluted tubule cells (PCT) and
glomerulus and (b) spleen with red pulp (RP) and white pulp (WP).
2.8.1: Gold nanoparticle-salt interaction assay. TEGylated nanoparticles were mixed in a 1:1 ratio with
either KCl or CaCl2 (0.1 molal final concentration) and left for five minutes to study interactions between
TEG nanoparticles and common serum salts via observing electrophoretic mobility differences.
A
volume of 4 µL of these solutions was mixed with 8 µL of LG buffer and 1.3 µL of 10x PBS (1x final
concentration), subjected to a current in a 4-20% Tris/Glycine gel, and this gel was scanned using an
Epson Scanner.
2.8.2: Gold nanoparticle-pH interaction assay. TEG nanoparticles were mixed in a 1:1 ratio with acid or
base creating aliquots with a final pH ranging from 1 to 14 with 1.0 pH increments to measure the effect
of pH on nanoparticle stability as visualized by electrophoretic mobility differences. After leaving these
samples overnight, 8 µL of LG buffer and 1.3 µL of 10x PBS (1x final concentration) were added to 4 µL
6
of sample and the aliquots were subjected to a current in a 4-20% Tris/Glycine gel. The gel was scanned
using an Epson Scanner.
2.8.3: Gold nanoparticle-GSH ligand exchange assay. TEG nanoparticles were mixed in a 1:1 ratio with
GSH dissolved in water to yield aliquots with a final GSH concentration of 0.1 mM (low concentration),
1 mM (low cellular concentration), 5 mM (medium cellular concentration), 10 mM (high cellular
concentration), 20 mM (high concentration), and 100 mM (very high concentration) to measure the
interactions between GSH and TEG nanoparticles per electrophoretic mobility variation. 14 The samples
were left to sit on a bench for one hour after which 8 µL of LG buffer and 1.3 µL of 10x PBS (final
concentration 1x PBS) was added to 4 µL of sample. The samples were then mixed, subjected to a
current in a 4-20% Tris/Glycine gel, and the gel was scanned using an Epson Scanner.
2.8.4: Assay for gold nanoparticle-BSA interactions following ligand exchange. TEG particles were
mixed in a 1:1 ratio with GSH (5 mM final concentration [medium cellular concentration]) and left for
one hour.14
The solution was then mixed 1:1 with serum levels of bovine serum albumin (final
concentration 38 mg/mL) and left for an hour to measure the interaction of serum proteins with ligandexchanged nanoparticles via observation of electrophoretic mobility variation.15 A volume of 4 µL of the
mixture was then mixed with 8 µL of LG buffer and 1.3 µL of 10x PBS (1x final concentration). The
sample along with nanoparticle controls (unaltered and ligand-exchanged particles) was subjected to a
current in a 4-20% Tris/Glycine gel, which was scanned using an Epson Scanner.
2.9: Mass spectrometric analysis of mouse organs. Inductively-coupled plasma mass spectrometry (ICPMS) was used to determine the amount of gold within mouse kidneys, spleens, and livers. 5 Samples were
(a) digested in undiluted nitric acid and 30% hydrogen peroxide, (b) diluted 1:160 in 2% nitric acid,
0.5% HCl, (c) injected into the equipment, (d) had their gold content determined by comparing mass
spectrometric counts of the samples to standards (1 ppb, 9.8 ppb, 24.3 ppb, 48.72 ppb, 101 ppb, 255 ppb),
(e) and had their gold content per gram of tissue measured by dividing the calculated concentration by the
sample mass and correcting for the 1:160 dilution.
7
2.10: Statistical analyses. Comparison of half-life between the different types of nanoparticles and
between batches of nanoparticles was determined using one-way ANOVA with an alpha value of 0.01
using the program JMP 9, which is available from the SAS Institute. Unless otherwise specified,
numerical data is presented as: Mean ± Standard Error.
3. Results
3.1: Gel electrophoretic assessment of gold nanoparticles. Gel electrophoresis revealed predominantly
mono-dispersed samples despite particle age (Figure 3a-b).
TEG gold nanoparticles migrated
significantly less than GSH particles (Figure 3a-b). The band representing GSH particles became more
diffuse and migrated less following 2.5 years in storage with TEG particles stored for six months lacking
such change (Figure 3a), suggesting GSH particles aggregated during storage as gels of samples closer in
age (less than one month age separation) yielded little variation in electrophoretic mobility (Figure 3b).
8
(a)
(b)
(g)
(h)
Core diameter: 3.40 nm ±
0.73 nm [n=1009]
Core diameter: 3.16 nm ±
0.60 nm [n=1003]
(c)
(e)
(d)
(f)
(i)
(j)
Core diameter: 5.11 nm ±
0.50 nm [n=1046]
Core diameter: 4.41 nm ±
0.78 nm [n=1050]
Figure 3. Gel of GSH and TEG gold nanoparticles with (a) different aged preparations in each well (age
difference > 6 months) and (b) similar aged preparations in each well (age difference < 1 month). TEM
images of (c) GSH nanoparticles and (d) TEG nanoparticles. Column chromatography histogram of (e)
GSH nanoparticles and (f) TEG nanoparticles. Histogram of (g) GSH nanoparticle core diameters for the
particles used in the first trial of experiments with their mean core diameter (Mean ± SD), (h) GSH
nanoparticle core diameters for the particles used in the second trial of experiments with their mean core
diameter (Mean ± SD), (i) TEG nanoparticle core diameters for the particles used in the first trial of
9
experiments with their mean core diameter (Mean ± SD), (j) TEG nanoparticle core diameters for the
particles used in the second trial of experiments with their mean core diameter (Mean ± SD).
3.2: Determination of gold nanoparticle core diameter. The following results are reported in terms of
mean ± standard deviation. TEM analysis showed that the GSH and TEG nanoparticles employed in my
experiments had significantly different core diameters (3.40 nm ± 0.73 nm [n=1009] and 5.11 nm ±
0.50 nm [n=1046], respectively; p < 0.01) (Figure 3c-d, g-j). Core diameter did not appear to greatly
vary between syntheses as results for a second batch of nanoparticles were within a standard deviation of
the other synthesis with the batch of GSH particles used in repeat trials having a core diameter of 3.16 nm
± 0.60 nm [n=1003] and the batch of TEG particles used in repeat trials having a core diameter of
4.41 nm ± 0.78 nm [n=1050] (Figure 3g-j).
3.3: Determination of gold nanoparticle hydrodynamic radius. Column analysis yielded approximate
hydrodynamic radii. Smaller fraction numbers (around 20) indicated larger hydrodynamic radii and
larger fraction numbers (around 49) indicated smaller hydrodynamic radii. GSH particles eluted at
fraction 36.5 with a second batch eluting at 35.8, suggesting a fairly reproducible synthesis and a particle
size slightly smaller than ovalbumin, a small protein with a molecular weight of 45.7 kD (Figure 3e).16
TEG particles eluted at fraction 33.4 and at fraction 34.2 for a second batch, suggesting once again a
reproducible synthesis and a size slightly smaller than bovine serum albumin (BSA), a large protein with
a molecular weight of 66.5 kD (Figure 3f).17 All column results revealed curves with tight peaks
suggesting fairly mono-dispersed samples, although there was a greater spread for GSH particles
compared to TEG particles suggesting lesser mono-dispersity (Figure 3e-f).
3.4: Determination of nanoparticle half-life in wild-type mice. It was found that the half-life of GSH and
TEG gold nanoparticles, as measured by semi-logarithmic trend-lines produced from beta phase timepoints (the period in the pharmacokinetic curve when a material is being metabolized and excreted
following distribution throughout the body), were 11.6 min ± 1.8 min (n = 4) and 67 min ± 3.5 min
(n = 5), respectively (Figure 4).13 The approximate lengths of the alpha phase for these particles (the
length of time for the material to distribute throughout the body and penetrate tissues) were 10 and 115
10
minutes, respectively (Figure 4).13 Note that the alpha phase length was greater than particle half-life for
TEG particles, but not for GSH nanoparticles.13 Following these experiments, fresh batches of GSH and
TEG nanoparticles were made to determine reproducibility. The half-life of the new GSH particles was
8.5 min ± 0.5 min (n = 3), which was not statistically different from the other synthesis (p = 0.72 > 0.01).
The half-life for the new TEG particles was 85 min ± 9.9 min (n = 3), which was also not statistically
different from the other synthesis (p = 0.04 > 0.01). Analysis of samples taken from animals injected
with these different batches of particles yielded an alpha phase length of 10 and 100 minutes, respectively
(data not shown).13
(a)
(b)
(c)
(d)
Figure 4. (a) One-way ANOVA diagram of nanoparticle half-lives with a Student’s T test. Splen refers
to splenectomized animals. (b) One-way ANOVA half-life statistics for each nanoparticle with Student’s
11
T test. Pharmacokinetic curves of (c) GSH and (d) TEG particles illustrating approximate alpha and beta
phases.13
3.5: Nanoparticle half-life determination in splenectomized mice. It was observed that TEG nanoparticles
accumulated in the spleen (Figure 6), so mice were splenectomized to investigate the organ’s role in
serum half-life. It was found that the half-lives of GSH and TEG particles in splenectomized animals
were 15.0 min ± 2.8 min (n = 2) and 66 min ± 4 min (n = 3), respectively, which were not statistically
different from either batch of GSH or TEG particles in wild type animals (p = 0.54 > 0.01 when these
GSH particle results were compared to the GSH particle batch with a half-life of 8.5 minutes and
p = 0.05 > 0.01 when these TEG particle results were compared to the TEG particle batch with a half-life
of 85 minutes) (Figure 4a-b).
When these experiments were repeated, they once again yielded
insignificant differences in half-life versus non-splenectomized animals with fresh GSH particles yielding
a half-life of 14.5 min ± 1.7 min (n = 2; p = 0.57 > 0.01 when compared to the GSH particles with a halflife of 8.5 minutes) and TEG particles yielding a half-life of 78 min ± 14 min (n = 3; p = 0.45 > 0.01
when compared to the TEG particle batch with a half-life of 85 minutes) (Figure 4a-b).
3.6.1: Tissue distribution of GSH particles. Kidney tissue sections from multiple animals injected with
multiple batches of nanoparticles suggest extensive renal uptake of GSH nanoparticles in proximal
convoluted tubule cells (PCTs) as visualized by dark punctuates (Figure 5a-c). This was accompanied
with excretion of black urine (suggesting nanoparticle presence). Splenic tissue sections suggested
moderate splenic uptake in both red and white pulp as visualized by dark punctuate (Figure 5d-f).18
There was no observed nanoparticle uptake in the liver (data not shown).
12
(a)
(b)
(c)
(d)
(e)
(f)
Figure 5. Thick kidney section of mouse injected with GSH nanoparticles (a) stained with toluidine blue,
100x. (b) stained with toluidine blue, 400x. (c) 400x. (d) stained with toluidine blue, 100x. (e) stained
with toluidine blue, 400x. (f) 400x.
3.6.2: Tissue distribution of TEG particles. Kidney tissue sections from multiple animals injected with
multiple batches of TEG particles suggest that this organ also takes up TEG gold nanoparticles, albeit less
so than GSH nanoparticles (Figure 6a-c). There was excretion of uncolored urine, suggesting a great
amount of particle re-uptake.
Analysis of splenic tissue suggests widespread accumulation of the
particles throughout the organ in both red and white pulp as visualized by dark punctuate (Figure 6).18
Liver tissue revealed no observable uptake (not shown).
13
(a)
(b)
(c)
(d)
(f)
Figure 6. Thick kidney section of
mouse injected with TEG particles
(a) stained with toluidine blue, 100x.
(b) stained with toluidine blue, 400x.
(c) 400x. (d) stained with toluidine
blue, 100x. (e) 100x.
(e)
(g)
3.6.3: Tissue distribution of nanoparticles in splenectomized animals.
(f) stained
with toluidine blue, 400x. (g) 400x.
Thick kidney sections from
animals injected with GSH and TEG particles revealed no observable difference in nanoparticle tubular
uptake between wild type and splenectomized animals for GSH particles, although they reveal more
observable uptake for TEG particles (Figure 7). As with wild-type animals, there was no visible
nanoparticle uptake by the liver (data not shown).
14
(a)
(b)
(c)
(d)
(e)
(f)
Figure 7. Thick kidney section of splenectomized mouse injected with (a) GSH particles stained with
toluidine blue, 100x. (b) GSH particles stained with toluidine blue, 400x. (c) GSH particles, 400x. (d)
TEG particles stained with toluidine blue, 100x. (e) TEG particles stained with toluidine blue, 400x. (f)
TTEG particles, 400x.
3.7: Gel electrophoretic analysis of mouse plasma following nanoparticle injection. Plasma samples from
mice injected with coated nanoparticles revealed no significant change in GSH particle migration while
circulating, although there was a time-based increase in electrophoretic mobility for TEG particles
(Figure 8a-b). This change was hypothesized to be due to ligand exchange reactions with intracellular
GSH as this was the only apparent means of increasing nanoparticle charge (column chromatography
refutes changes in nanoparticle size and gel electrophoresis results suggest the change is not due to ion
15
interactions and that the concentration of GSH in serum [roughly 64.5 µM] was insufficient to induce an
electrophoretic migration change) (Figures 8-9).19-22
Only intracellular concentrations of GSH
(concentrations greater than 1 mM) were found to trigger faster nanoparticle migration (Figure 8c).14,19
These ligand-exchanged particles were subsequently found to be sensitive to protein adsorption as mixing
them with a serum concentration of BSA (38 mg/mL) decreased electrophoretic mobility (Figure 8c).15,19
There was decreased band intensity for all samples over time while flowing through circulation, which
was a product of their filtration from serum (Figure 8a-b). There was also slowed migration of the
bromophenol blue marker in samples mixed with serum; this was a product of the dye’s binding with
serum albumin (Figure 8a-b).23 Lastly, there was loss of lower and higher molecular weight GSH
particles over time; this was likely due to rapid glomerular filtration of smaller particles and splenic
uptake of larger material, as indicated by smaller material being present in the lane running nanoparticlecontaining mouse urine from the same animal and spleen sections revealing nanoparticle aggregates
(Figures 5, 7-8).1,24
16
(a)
(b)
(d)
(c)
Figure 8. (a) Gel of plasma 3, 6, 10, 15, and 20 minutes following injection of GSH nanoparticles. NP
refers to a nanoparticle sample never exposed to plasma and U refers to a urine sample. (b) Gel of plasma
5, 166, 333, and 500 minutes following injection of TEG nanoparticles. (c) Gel of TEG particles mixed
with GSH for 1 hour (low concentration [0.1 mM], low cellular concentration [1 mM], medium cellular
17
concentration [5 mM], high cellular concentration [10 mM], high concentration [20 mM], very high
concentration [100 mM]) and TEG particles mixed with 5 mM GSH for one hour and a serum
concentration of albumin (38 mg/mL) for one hour.14-5 (d) Column data of TEG particles, TEG particles
mixed with wild-type mouse plasma, and TEG particles in plasma 166 min following injection.
3.7: Column analysis of mouse plasma following nanoparticle injection.
Column chromatographic
analysis of TEG particles in saline, mixed with wild-type mouse plasma, and in plasma 166 minutes
following nanoparticle injection suggested insignificant nanoparticle-macromolecule interactions based
on little variation in both elution fraction and spread between these three samples. While there were some
peaks ahead of the large nanoparticle peak in the third sample, these were likely unbound plasma proteins
due to their high molecular weight, separation from the nanoparticle peak, and gel electrophoresis of the
sample showing solely electrophoretic acceleration suggesting that nanoparticle size did not increase
(Figure 8b,d).
3.8: ICP-MS analysis of mouse kidney, spleen, and liver. Of the three organs studied, variation in
nanoparticle uptake based on coat or splenectomy was only evident for kidney tissue; there was greater
nanoparticle uptake for GSH particles and non-splenectomized animals.
There was significant
nanoparticle uptake in the kidney of both types of nanoparticles with significantly more GSH particle
uptake compared to TEG particle uptake (p < 0.01) and a significant decrease in uptake of GSH particles
following splenectomy (p < 0.01 for GSH particles, p = 0.06 > 0.01 for TEG particles) (Figure 9). There
was also significant nanoparticle uptake in the spleen for both types of nanoparticles with no significant
difference in uptake of GSH or TEG particles (p = 0.82 > 0.01) (Figure 9). There was also significant
nanoparticle uptake in the liver for both types of nanoparticles with no difference in uptake based on
particle coat (p = 0.90 > 0.01) or status of splenectomy (p = 0.39 > 0.01 for GSH and p = 0.93 > 0.01 for
TEG) (Figure 9).
18
(a)
(b)
(c)
Figure 9. ICP-MS results of (a) kidney, (b) spleen, and (c)
liver with standard error.
4. Discussion
4.1: GSH and TEG nanoparticle syntheses produce reproducible samples. There was no significant
variation in nanoparticle half-life, electrophoretic migration, core diameter, or hydrodynamic radius
between newly made batches of GSH and TEG particles (Figures 3-4). This suggests that their protocols
are producing comparable samples; however, there are differences. GSH particles migrated significantly
further than TEG particles (Figure 3). This is likely due to variation between solvent-exposed chemical
groups where TEG has solely a free hydroxyl group (neutral at physiological and electrophoretic pH)
whereas GSH has two negatively charged carboxyl groups and a free amine group (producing a
19
negatively-charged compound at physiological pH).9 The relative standard deviation for GSH particle
core diameter was also larger than that of TEG particles (21% and 19% versus 9.8% and 18%) (Figure 3).
Further, the relative standard error of half-life for GSH particles in wild-type animals was slightly greater
than and less consistent than TEG particles (10% and 4% versus 5% and 7%) (Figure 4). Lastly, TEG
particles were more monodispersed than GSH particles based on column chromatography (Figure 3).
These results suggest that TEG particles have a more reproducible synthesis than GSH particles. As
nanoparticles can be coated with a glycol and another compound, like the chemotherapeutic agent
doxorubicin, consistency and monodispersity of nanomaterial is valuable because it can maximize
filtration to a specific cell, like a cancer cell in doxorubicin’s case.1
4.2: TEG particles have a longer half-life than GSH particles. GSH nanoparticles had a significantly
shorter half-life than TEG nanoparticles (11.6 minutes compared to 71 minutes) showing the ability of
glycol coats to increase serum half-life (Figure 4). Column data revealed that TEG particles had
hydrodynamic radii comparable to BSA, which is subject to limited glomerular filtration24, and GSH
particles had hydrodynamic radii similar to ovalbumin, a molecule more readily filtered by the kidney’s
glomerulus due to its smaller size (Figure 3).16-7,24 This significant difference in particle half-life is likely
due to the smaller GSH particles more readily passing through the glomerular basement membrane,
leading to more rapid filtration versus the larger TEG particles.24 As there was significant renal uptake of
GSH and TEG particles for both splenectomized and nonsplenectomized animals according to mass
spectrometry (Figure 9) and there was no change in particle half-life following splenectomy (Figure 4),
this suggests that the kidney is a major filter of these nanoparticles and that hydrodynamic diameter is a
determining factor of nanoparticle half-life with larger particles having a longer half-life when controlling
for protein adsorption (Figures 6-7).24
4.3: TEG particles react with a cellular-based compound. The GSH particles were fairly inert in serum
as gel electrophoresis revealed little migration change; however, TEG particles appeared to react with a
material based on their increasing electrophoretic mobility during circulation (Figure 8). Column data
indicated no significant interaction of TEG particles with macromolecules based on a seemingly
20
unchanged hydrodynamic radius 166 minutes following injection compared to hydrodynamic radius
before serum exposure. This suggests that the particles are relatively unreactive towards macromolecules
and stable in serum (Figure 8). The TEG particle alpha phase length (time for nanoparticles to distribute
throughout serum and permeate tissues) is longer than particle half-life, further suggesting their limited
interaction with proteins (Figure 4).9,13 This is likely a product of TEG conferring resistance to protein
adsorption that would limit particle permeability into tissues, making susceptibility to macrophage-based
filtration a significant moderator of their half-life.5,7-10,18 The change in electrophoretic mobility is thus
most likely due to interaction with small molecules.
Gel electrophoresis illustrated that neither
concentrated salts (potassium, sodium, calcium) nor physiological pH values elicited the observed change
(Figure 9).21 Although it has been found that both PEG and TEG particles can undergo ligand exchange
reactions (that would displace protective TEG groups that would increase probability of phagocytosis),
the concentration of common thiols in serum (GSH and cysteine) is too low to elicit this reaction (their
concentrations are roughly 68.1µM and 79.6µM, respectively) (Figure 8).9,19,22 Therefore, TEG particles
must undergo these ligand exchange reactions intracellularly where there is adequate concentration of
GSH.
4.4: TEG particles are taken up by kidney proximal convoluted tubule cells following glomerular
filtration. Both types of nanoparticles accumulated in kidney PCT cells as they were able to filter through
the glomerular basement membrane due to a hydrodynamic radius that is small enough to permit filtration
(GSH particles filtered more quickly due to their significantly smaller size compared to TEG particles as
supported by thick sections, mass spectrometry, and urine samples) (Figures 5-6, 9).16-7,24 Upon filtering
into the glomerulus and moving into the PCT, the particles were absorbed by tubule cells (demonstrated
by kidney thick sections revealing extensive presence of nanoparticles in PCT cells) and returned to
circulation to again filter through the kidney or be collected by metallophilic macrophages in the spleen or
liver (Figures 5-6).5,18,24 GSH particles were observed in the urine, which was expected because their
small hydrodynamic radius permits more rapid filtration into the kidney, leading the particles to exceed
the transport maximum of reabsorption-related proteins on PCT cell membranes.24 This would permit
21
them to pass into more distant tubules and ultimately filter into the urine (filtration into distal convoluted
tubule cells was not investigated).24 TEG particles were likely not observed in the urine because they
would filter through the kidney’s glomerular basement membrane at a significantly slower rate due to
their larger hydrodynamic radius, thus not exceeding the transport maximum of PCT cell membrane
proteins.16-7,24 This moderated filtration could explain the decreased TEG particle presence observed on
thick sections compared to GSH particles and mass spectrometry showing a lower concentration of TEG
particles in the kidney compared to GSH particles (Figures 5-6, 9).20,24 There was a decrease in renal
gold concentration following splenectomy; however, at this time, this decrease cannot be attributed to the
procedure as sham surgeries (performing the operation, but not removing the spleen) were not conducted
(Figure 9). The decrease could be due to homeostatic imbalances produced by the surgery rather than the
splenectomy because while literature suggests that the spleen has a role in regulating glomerular basement
membrane filtration in rats, no papers could be found documenting the effect of splenectomy on this
measurement.25
4.5: TEG particles are modified in kidney proximal convoluted tubule cells. Gel electrophoresis and
column chromatography suggest that TEG particles are being modified in PCT cells, most likely by being
subjected to ligand exchange reactions with GSH.14,19 This is the most likely location of this reaction as
the trend was demonstrated comparably in both splenectomized and non-splenectomized animals, there
was evidence of significant renal uptake in both of these animal groups, and of known nanoparticle
uptake mechanisms, this is the only path that would return nanoparticles to circulation (the spleen and
liver use macrophages to phagocytose the particles that would limit the probability they would return to
circulation) (Figures 5-7, 9).1,5,18-9 Intracellular concentrations of GSH (which are generally between 1
mM and 10 mM) were found to be sufficient to induce this reaction to the extent seen in plasma samples
as observed by a gel demonstrating comparable increases in particle migration at these concentrations of
GSH.14 This exchange reaction would facilitate nonspecific protein adsorption in serum with proteins
such as albumin or macrophage receptor proteins that could facilitate phagocytosis (Figure 8c).1,19 While
particles coated with GSH are documented as being fairly resistant to protein adsorption, which is
22
supported by gel electrophoresis experiments (Figure 8), this ligand exchange reaction perhaps
introduces novel electron repulsions between TEG oxygen groups and GSH carboxyl groups on the
nanoparticle surface, making space for proteins to interact, as depicted in Larson et al’s work. 11,19
Although column chromatography showed no great change in TEG particle size, column chromatography
is limited in studying nanoparticle-protein interactions because nanoparticles are known to form transient
protein interactions that are very possibly broken during column traversal due to induced separation,
which would suggest no interaction.26 This data thus suggests that TEGylated particles are forming
transient interactions with serum proteins that could facilitate phagocytosis by macrophages in the spleen
and liver.1,26
4.6: TEG particles are taken up in the spleen following their adsorption of serum proteins. Splenic tissue
sections suggest little uptake of GSH nanoparticles and significant uptake of TEG nanoparticles (Figures
5-6). Mass spectrometric analysis details an insignificant difference in spleen gold concentration between
the particles, although comparison to other tissues reveals that a greater proportion of TEG particles are
filtered by the spleen versus GSH particles, perhaps explaining the variation in particle visualization
(Figures 5-6, 9). GSH particles were likely less visibly taken up by the spleen and their half-life not
altered following splenectomy due to their rapid filtration through the renal glomerulus, smaller overall
size that limits splenic macrophage uptake1, and limited reuptake by PCT cells (which would further
expose them to serum proteins upon returning to circulation and facilitate protein adsorption that could
increase hydrodynamic diameter and trigger preferential splenic filtration) (Figures 4-5).6,20,24 TEG
particles were likely more visibly taken up by the spleen, which is not their primary filter as their half-life
did not significantly vary following splenectomy, due to their slower filtration through the glomerulus
that would permit their observed apparent complete reuptake by PCT cells that would return them to
circulation (Figure 6).18,20,24 Their increased exposure and subsequent binding with serum-based proteins
upon return to circulation would then facilitate phagocytosis in the spleen as it has been found that
nanoparticle binding with serum proteins, such as immunoglobulins and complement factors, can
facilitate particle uptake by macrophages in the spleen due to a greater hydrodynamic radius. 1,19,27 This is
23
demonstrated by the larger aggregates of particles depicted on spleen sections (Figure 6) that suggest
multiple particles can adsorb to the same serum proteins, increasing probability of forming an aggregate
large enough to trigger preferential splenic filtration.19 The particles taken up by the spleen had no
distinguishable target with varying sized aggregates found within both the red and white pulp (Figures 56).18 Particles collected in the red pulp were likely taken up by macrophages in the region’s reticular
meshwork18; previous work has found that injections of thorium dioxide and hemosiderin, an iron storage
complex, are taken up by macrophages in this region, suggesting metallophilic characteristics. 28 Particles
collected in the white pulp were likely taken up by the well-described metallophilic macrophages present
there.18
4.7: TEG particles are greatly taken up by liver-based metallophilic macrophages. It is known that liver
Kupffer cells (metallophilic macrophages) can take up gold nanoparticles. 5 Thick sections of this tissue
from experimental mice revealed no significant observed variation between animals injected with saline
and animals injected with both of my types of nanoparticles (data not shown).
However, mass
spectrometry revealed extensive uptake of both types of gold nanoparticles in this organ making it one of
their dominant filters. There was no significant difference in filtration based on nanoparticle coat, as
expected according to literature documenting extensive liver uptake of nanoparticles with core diameters
less than 10nm, which characterizes the majority of my GSH and TEG nanoparticles (Figure 9).1 A
greater proportion of TEG particles were taken up by this organ compared to GSH particles, likely due to
their slower filtration through the glomerular basement membrane and what appeared to be complete
uptake by renal PCT cells following filtration. This would expose them further to hepatic and splenic
macrophages as well as serum proteins whose adsorption could further increase probability of hepatic and
splenic filtration (Figure 9).1,18,20,24
5. Conclusions:
These results suggest that a glycol coat, tested using TEG as a coating agent for gold
nanoparticles, confers an extended half-life and resistance to non-specific protein adsorption. A GSH
coat also appears to confer resistance to non-specific protein adsorption, but does not provide the
24
extended half-life of a glycol coat likely due to the particles having greater permeability through the
glomerular basement membrane, effectively limiting reuptake by PCT cells. The syntheses I used to
make these particles were shown to be reproducible with TEG particles forming more mono-dispersed
samples according to column chromatography. Both particles were greatly filtered by the liver and
glomerulus to be collected in Kupffer cells and PCT cells as supported by ICP-MS and thick section
analysis. TEG particles were observed to increase in charge over time, which was likely due to their
conduction of ligand exchange reactions with cellular based thiols, such as GSH, in PCT cells. This
reaction would facilitate their phagocytosis by splenic and hepatic macrophages following transient
adsorption of serum proteins that could induce aggregation of nanoparticles, as visualized in spleen tissue.
These experiments indicate that TEG and GSH are useful coating agents for gold nanoparticles with TEG
conferring an extended half-life and its nanoparticle synthesis making fairly mono-dispersed particles,
both of which could be useful for pharmacological applications. However, TEG particle filtration must
be further investigated to assess toxicity as no removal mechanism could be identified that removed these
particles from the body. It is already known that PEG nanoparticles can induce negative physiological
consequences (including acute inflammation and apoptosis in the liver)5; it must be determined if TEG
particles have similar cytotoxic effects so one may decide if the benefits conferred by nanoparticledelivered drug outweighs costs associated with cytotoxicity.
6. Abbreviations: BSA = Bovine serum albumin. GSH = Glutathione. ICP-MS = Inductively coupled
plasma mass spectrometry. PCT = Proximal convoluted tubule. PEG = Poly-ethylene glycol. RP = Red
pulp. Splen = Splenectomy. TEG = Tetra-ethylene glycol. WP = White pulp.
7. Acknowledgements: This work was made possible by a University of North Carolina Summer
Undergraduate Research Fellowship, Honors Carolina, grants from the National Institutes of Health
(HL49277 and DK080302), Robert Bagnell and Vicky Madden for their assistance with electron and light
microscopy, Dr. Daniel Kenan for his assistance in histological section cellular identification, Dr. Peter
Cable for his assistance in performing ICP-MS, Dr. Marlon Lawrence for his assistance in nanoparticle
25
synthesis, Dr. Feng Li for her assistance with mouse experiments, Dr. Kelly Hogan for her role as a
research advisor, and Dr. Oliver Smithies for his role as a mentor.
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28
9. Supplemental Materials:
(a)
(b)
29
Figure 10.
Gel of TEG particles mixed with (a) potassium or calcium chloride (0.1 molal final
concentration) and (b) acid/base to a final pH of 1 to 14.
(a)
(b)
30
Figure 11. Gel of splenectomized animal plasma (a) 3, 5, 10, 15, 20, and 30 minutes following injection
of GSH particles and (b) 25, 166, 333, and 500 minutes following injection of TEG particles. NP stands
for nanoparticle sample never exposed to plasma and U refers to a urine sample.
31